THERAPEUTIC AGENTS TARGETING THE NCCa-ATP CHANNEL AND METHODS OF USE THEREOF

ABSTRACT

The present invention is directed to therapeutic compositions targeting the NCCa-ATP channel of an astrocyte, neuron or capillary endothelial cell and methods of using same. More specifically, agonists and antagonists of the NCCa-ATP channel are contemplated. The therapeutic compositions are used to treat cancer, more specifically, a metastatic brain tumor, wherein a tumor-brain barrier is present. Such treatments are contemplated in combination with conventional anti-cancer therapies. Alternatively, the compositions are used to prevent cell death and to treat cerebral edema that result from ischemia, due to interruption of blood flow, to tissue trauma or to increased tissue pressure.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 11/574,793 filed on Oct. 30, 2008, which is anational phase application under 35 U.S.C. § 371 that claims priority toInternational Application No. PCT/US05/26455 filed Jul. 25, 2005, whichclaims priority to U.S. Provisional Application No. 60/610,758 filedSep. 18, 2004, all of which are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. NS048260awarded by the National Institutes of Health and under Grant NumberNEUC-005-01F awarded by the United States Department of VeteransAffairs. The government has certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“Seq_Lst_UOMD_P0006US_C4.TXT”, which is 3 KB and filed herewith byelectronic submission and is incorporated by reference herein.

TECHNICAL FIELD

The present invention is directed to fields of cell biology, physiologyand medicine. More specifically, the present invention addresses novelmethods of treating a patient comprising administering a therapeuticcompound that targets a unique non-selective cation channel activated byintracellular calcium and blocked by intracellular ATP (NC_(Ca-ATP)channel). In specific embodiments, the therapeutic compound is anagonist, and uses thereof in therapies, such as cancer therapies,benefiting from death of neuronal cells. In other specific embodiments,the therapeutic compound is an antagonist, and uses thereof intherapies, such as treatment of cerebral ischemia or edema, benefitingfrom blocking and/or inhibiting the NC_(Ca-ATP) channel. Compositionscomprising agonists and/or antagonists of the NC_(Ca-ATP) channel arealso contemplated.

BACKGROUND OF THE INVENTION I. NC_(Ca-ATP) Channel

A unique non-selective monovalent cationic ATP-sensitive channel(NC_(Ca-ATP) channel) was identified first in native reactive astrocytes(NRAs) and later, as described herein, in neurons and capillaryendothelial cells after stroke or traumatic brain injury (See,International application WO 03/079987 to Simard et al., and Chen andSimard, 2001, each incorporated by reference herein in its entirety).The NC_(Ca-ATP) channel is thought to be a heteromultimer structurecomprised of sulfonylurea receptor type 1 (SUR1) regulatory subunits andpore-forming subunits, similar to the K_(ATP) channel in pancreatic βcells (Chen et al., 2003). The pore-forming subunits of the NC_(Ca-ATP)channel remain uncharacterized.

SUR imparts sensitivity to antidiabetic sulfonylureas such asglibenclamide and tolbutamide, and is responsible for activation by achemically diverse group of agents termed “K⁺ channel openers” such asdiazoxide, pinacidil and cromakalin (Aguilar-Bryan et al., 1995; Inagakiet al., 1996; Isomoto et al., 1996; Nichols et al., 1996; Shyng et al.,1997). In various tissues, molecularly distinct SURs are coupled todistinct pore-forming subunits to form different K_(ATP) channels withdistinguishable physiological and pharmacological characteristics. TheK_(ATP) channel in pancreatic β cells is formed from SUR1 linked withKir6.2, whereas the cardiac and smooth muscle K_(ATP) channels areformed from SUR2A and SUR2B linked with Kir6.2 and Kir6.1, respectively(Fujita et al., 2000). Despite being made up of distinctly differentpore-forming subunits, the NC_(Ca-ATP) channel is also sensitive tosulfonylurea compounds.

Also, unlike the K_(ATP) channel, the NC_(Ca-ATP) channel conductssodium ions, potassium ions, cesium ions and other monovalent cationswith near equal facility (Chen and Simard, 2001) suggesting further thatthe characterization, and consequently the affinity to certaincompounds, of the NC_(Ca-ATP) channel differs from the K_(ATP) channel.

Other nonselective cation channels that are activated by intracellularCa²⁺ and inhibited by intracellular ATP have been identified but not inastrocytes. Further, the NC_(Ca-ATP) channel expressed and found inastrocytes differs physiologically from the other channels with respectto calcium sensitivity and adenine nucleotide sensitivity (Chen et al.,2001).

II. Gliotic Capsule

The gliotic capsule that forms around a “foreign body” in the brain isan important, albeit neglected, biological system. On the one hand, thegliotic capsule represents the response of the brain to an injuriousstimulus—an attempt by the brain to wall off, isolate, dispose of, andotherwise protect itself from the foreign body. On the other hand, thegliotic capsule forms a potentially harmful mass of tissue from whichoriginates edema fluid that contributes to brain swelling, and whoseconstituent cells undergo cytotoxic edema, which adds further to brainswelling. Also, the gliotic capsule protects foreign cells fromimmunologic surveillance.

The essential elements involved in formation of a gliotic capsule appearto be uniform in many types of CNS pathology, be it a traumaticallyimplanted foreign body, a metastatic tumor, a brain abscess, orinfarcted necrotic tissue following a stroke. First, microglia andastrocytes become activated near the site of injury, with large,stellate-shaped GFAP-positive reactive astrocytes forming the mostprominent cellular component of the response. Secondly, the foreignnature of the entity is recognized, and the response is initiated tosurround and contain it. Although the concept of “foreign body”encompasses a large variety of pathological conditions, the responses inmost cases bear a great deal of similarity to one another.

The interface between the foreign body and the gliotic capsule, referredto as the inner zone of the gliotic capsule, appears to be of greatimportance in determining the overall response to injury.

Despite the overall benefits, the gliotic capsule forms a potentiallyharmful mass of tissue that contributes to brain swelling and masseffect, and that may shelter foreign cells from surveillance by theimmune system. Applicants are the first to determine that, in a varietypathological conditions in both rats and humans, reactive astrocytes (R1astrocytes) in the inner zone of the gliotic capsule express a novelSUR1-regulated cation channel, the NC_(Ca-ATP) channel, and that thischannel directly controls cell viability: opening the channel isassociated with necrotic cell death and closing the channel isassociated with protection from cell death induced by energy (ATP)depletion.

III. Cancer

Brain metastasis is an important cause of morbidity and mortality incancer patients. Because most of these patients die of systemic disease,the primary therapeutic goal is often simply to improve the quality oflife. Conventional therapy for brain metastases is usually whole-brainirradiation. Chemotherapy may result in regression of brain metastasesin chemosensitive tumors, but overall, results of adjunctive therapyincluding chemotherapy and immunotherapy are disappointing.

The most widely recognized “barrier” that isolates brain metastases isthe blood-brain barrier (BBB). In addition, the gliotic capsule thatforms around the metastasis forms a “tumor-brain barrier” (TBB) thatalso isolates and protects a metastatic tumor. Unlike primaryCNS-derived tumors such as glioblastoma, metastatic cancers of the braininduce a significant astrocytic reaction, resulting in formation of agliotic capsule. The gliotic capsule that forms around a metastatictumor represents the response of the brain to an injurious stimulus—anattempt by the brain to wall off, isolate, dispose of, and otherwiseprotect itself from the metastatic tumor. Importantly, however, thegliotic capsule also functions as a barrier that protects the metastatictumor from immunologic surveillance and therapeutic targeting.

Successful immunotherapy and chemotherapy for metastatic brain tumorsremains elusive. In general, the difficulty in treating these tumors isascribed to presence of the blood brain barrier (BBB), which is believedto prevent access of chemotherapeutic agents and immunological cells totumors located in the brain. However, much of the blood supply tometastatic tumors in the brain originates from vessels and capillarieslocated in the gliotic capsule that surrounds the tumor, and thesecapillaries, unlike those in brain per se, are fenestrated. The glioticcapsule itself that surrounds the tumor has an inner zone that ispopulated by R1 astrocytes that express tight junction proteins and thisinner zone is thought to form a barrier between tumor and brain. Thebarrier formed by R1 astrocytes is termed the tumor-brain barrier (TBB).

Monotherapies with chemotherapeutic agents tends not to be veryeffective because conventional chemotherapeutic agents tend not to reachportions of the CNS in effective amounts, primarily because of theblood-brain barrier (BBB). For example, etoposide and actinomycin D, twocommonly used oncology agents that inhibit topoisomerase II, fail tocross the blood-brain barrier in useful amounts.

As described herein, Applicants are the first to determine that theinner zone of the gliotic capsule is populated by R1 astrocytesexpressing the NC_(Ca-ATP) channel, and selectively killing theastrocytes expressing the NC_(Ca-ATP) channel disrupts the TBB, causingmigration of leukocytes across the TBB.

Other and further objects, features, and advantages will be apparentfrom the following description of the presently preferred embodiments ofthe invention, which are given for the purpose of disclosure.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a unique non-selective cation channelactivated by intracellular calcium and blocked by intracellular ATP(NC_(Ca-ATP) channel) that can be expressed in neuronal cells, neurogliacells (e.g., astrocyte, ependymal cell, oligodentrocyte and microglia)or neural endothelial cells (e.g., capillary endothelial cells) in whichthe cells have been or are exposed to a traumatic insult, for example,an acute neuronal insult (e.g., hypoxia, ischemia, cerebral edema orcell swelling), toxic compounds or metabolites, an acute injury, cancer,brain abscess, etc. More particularly, the present invention relates tothe regulation and/or modulation of this NC_(Ca-ATP) channel and how itsmodulation can be used to treat various diseases and/or conditions, forexample hyperproliferative diseases and acute neuronal insults (e.g.,stroke, an ischemic/hypoxic insult). Yet further, the present inventionrelates to the regulation and/or modulation of this NC_(Ca-ATP) channeland its role in maintaining or disrupting the integrity of the glioticcapsule. The modulation and/or regulation of the channel results fromadministration of an activator or agonist of the channel or anantagonist or inhibitor of the channel. Thus, depending upon thedisease, a composition (an antagonist or inhibitor) is administered toblock or inhibit the channel to prevent cell death, for example to treatcerebral edema that results from ischemia due to tissue trauma or toincreased tissue pressure. In these instances the channel is blocked toprevent or reduce or modulate depolarization of the cells. In the caseof cancer or other hyperproliferative diseases, it is desirable to openor activate the channel by administering an agonist or activatorcompound to cause cell depolarization resulting in cell death of thecancer cells or hyperproliferative cells.

The composition(s) of the present invention may be delivered alimentaryor parenterally. Examples of alimentary administration include, but arenot limited to orally, buccally, rectally, or sublingually. Parenteraladministration can include, but are not limited to intramuscularly,subcutaneously, intraperitoneally, intravenously, intratumorally,intraarterially, intraventricularly, intracavity, intravesical,intrathecal, or intrapleural. Other modes of administration may alsoinclude topically, mucosally, transdermally, direct injection into thebrain parenchyma.

An effective amount of an agonist or antagonist of NC_(Ca-ATP) channelthat may be administered to a cell includes a dose of about 0.0001 nM toabout 2000 μM. More specifically, doses of an agonist to be administeredare from about 0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM;about 0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5μM to about 2.0 μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about4.0 μM; about 4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about10 μM to about 50 μM; about 50 μM to about 100 μM; about 100 μM to about200 μM; about 200 μM to about 300 μM; about 300 μM to about 500 μM;about 500 μM to about 1000 μM; about 1000 μM to about 1500 μM and about1500 μM to about 2000 μM. Of course, all of these amounts are exemplary,and any amount in-between these points is also expected to be of use inthe invention.

An effective amount of an agonist and/or antagonist of the NC_(Ca-ATP)channel or related-compounds thereof as a treatment varies dependingupon the host treated and the particular mode of administration. In oneembodiment of the invention the dose range of the agonist and/orantagonist of the NC_(Ca-ATP) channel or related-compounds thereof willbe about 0.01 μg/kg body weight to about 20,000 μg/kg body weight. Theterm “body weight ” is applicable when an animal is being treated. Whenisolated cells are being treated, “body weight” as used herein shouldread to mean “total cell body weight”. The term “total body weight” maybe used to apply to both isolated cell and animal treatment. Allconcentrations and treatment levels are expressed as “body weight” orsimply “kg” in this application are also considered to cover theanalogous “total cell body weight” and “total body weight”concentrations. However, those of skill will recognize the utility of avariety of dosage range, for example, 0.01 μg/kg body weight to 20,000μg/kg body weight, 0.02 μg/kg body weight to 15,000 μg/kg body weight,0.03 μg/kg body weight to 10,000 μg/kg body weight, 0.04 μg/kg bodyweight to 5,000 μg/kg body weight, 0.05 μg/kg body weight to 2,500 μg/kgbody weight, 0.06 μg/kg body weight to 1,000 μg/kg body weight, 0.07μg/kg body weight to 500 μg/kg body weight, 0.08 μg/kg body weight to400 μg/kg body weight, 0.09 μg/kg body weight to 200 μg/kg body weightor 0.1 μg/kg body weight to 100 μg/kg body weight. Further, those ofskill will recognize that a variety of different dosage levels will beof use, for example, 0.0001 μg/kg, 0.0002 μg/kg, 0.0003 μg/kg, 0.0004μg/kg, 0.005 μg/kg, 0.0007 μg/kg, 0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0 μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/kg, 100 μg/kg, 120 μg/kg, 140 μg/kg,150 μg/kg, 160 μg/kg, 180 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 450 μg/kg,500 μg/kg, 550 μg/kg, 600 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 900μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, and/or30 mg/kg. Of course, all of these dosages are exemplary, and any dosagein-between these points is also expected to be of use in the invention.Any of the above dosage ranges or dosage levels may be employed for anagonist and/or antagonist of NC_(Ca-ATP) channel or related-compoundsthereof.

The NC_(Ca-ATP) channel is blocked by antagonists of type 1 sulfonylureareceptor (SUR1) and opened by SUR1 activators. More specifically, theantagonists of type 1 sulfonylurea receptor (SUR1) include blockers ofK_(ATP) channels and the SUR1 activators include activators of K_(ATP)channels. More specifically, the NC_(Ca-ATP) channel of the presentinvention has a single-channel conductance to potassium ion (K⁺) between20 and 50 pS. The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on thecytoplasmic side of the cell membrane in a physiological concentrationrange, where concentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP)channel is also inhibited by cytoplasmic ATP in a physiologicalconcentration range, where the concentration range is from 10⁻¹ to 10 M.The NC_(Ca-ATP) channel is also permeable to the following cations; K⁺,Cs⁺, Li⁺, Na⁺; to the extent that the permeability ratio between any twoof the cations is greater than 0.5 and less than 2.

Certain embodiments of the present invention comprise a method oftreating a hyperproliferative disease by administering to a subject anamount of a compound effective to activate a NC_(Ca-ATP) channel in aneuronal cell or a neuroglia cell or a neural endothelial cell or acombination thereof. The activation of the channel results in an influxof sodium ions (Na⁺) causing depolarization of the cell. The influx ofNa⁺ alters the osmotic gradient causing an influx of water into the cellwhich leads to cytotoxic edema ultimately resulting in necrotic celldeath.

The hyperproliferative disease is a tumor, for example, a benign ormalignant tumor. More specifically, the tumor is a neuroma or glioma.Still further, the tumor can originate from a primary brain tumor ormetastatic brain tumor. Gliomas can include, but are not limited toastocytoma, brain stem glioma, ependymomas, optic nerve glioma, andoligodendroglioma. The tumor may also be gliobastoma, medulloblastoma,papilloma of choroid plexus, metastases, meningioma, pituitary adenoma,Schwannoma, lymphoma, congenital tumors, neurosarcoma,neurofibromatosis, neuroblastoma, craniopharyngioma, pineal regiontumors or primitive neuroectodermal tumors.

The activator compound or agonist can be a type 1 sulfonylurea receptoragonist. For example, agonists that can be used in the present inventioninclude, but are not limited to agonist of SUR1, for example, diazoxide,pinacidil, P1075, and cromakalin. Other agonists can include, but arenot limited to diazoxide derivatives, for example3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC55-9216), 6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine1,1-dioxide (BPDZ 154),7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ73), 6-Chloro-3-isopropylamino-4 H-thieno[3,2-e]-1,2,4-thiadiazine1,1-dioxide (NNC 55-0118)4, 6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414),3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide(BPDZ 44),3-(1′,2′,2′-trimethylpropyl)amino-4H-pyrido(4,3-e)-1,2,4-thiadiazine1,1-dioxide (BPDZ 62), 3-(1′,2′,2′-trimethylpropyl)amine-4H-pyrido(2,3-e)-1,2,4-thiadiazine, 1,1-dioxide (BPDZ 79),2-alkyl-3-alkylamino-2H-benzo- and2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides,6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxidederivatives, 4-N-Substituted and -unsubstituted 3-alkyl- and3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides. Inaddition, other compounds, including 6-chloro-2-methylquinolin-4(1H)-one(HEI 713) and LN 533021, as well as the class of drugs,arylcyanoguanidines, are known activators or agonist of SUR1. Othercompounds that can be used include compounds known to activate K_(ATP)channels.

In further embodiments, the method comprises administering to thesubject an anti-cancer therapy in combination with the activatorcompound that activates or stimulates or opens the NC_(Ca-ATP) channel.The anti-cancer or anti-tumor therapy is chemotherapy, radiotherapy,immunotherapy, surgery or a combination thereof.

Another embodiment of the present invention comprises a method ofdisrupting the integrity of the tumor-brain barrier surrounding a tumorin the brain of a subject comprising administering to the subject acompound effective to activate a NC_(Ca-ATP) channel in a neuronal cell,or a neuroglia cell, a neural endothelial cell or a combination thereof.This method can further comprise administering to the subject ananti-cancer therapy, wherein the anti-cancer or anti-tumor therapy ischemotherapy, radiotherapy, immunotherapy, surgery or a combinationthereof.

Still further, another embodiment of the present invention comprises amethod of inducing cell death of a neuronal or a neurolgia cell or aneural endothelial cell comprising administering to the cell a compoundeffective to activate a NC_(Ca-ATP) channel in the cell. Activation ofthe NC_(Ca-ATP) channel results in an influx of sodium ions (Na⁺)causing depolarization of the cell. The influx of Na⁺ alters the osmoticgradient causing an influx of water into the cell which leads tocytotoxic edema ultimately resulting in necrotic cell death.

Yet further, another embodiment of the present invention comprises apharmaceutical composition comprising a thrombolytic agent (e.g., tissueplasminogen activator (tPA), urokinase, prourokinase, streptokinase,anistreplase, reteplase, tenecteplase), an anticoagulant or antiplatelet(e.g., aspirin, warfarin or coumadin), statins, diuretics, vasodilators,mannitol, diazoixde or similar compounds that stimulates or promotesischemic precondition or a pharmaceutically acceptable salt thereof anda compound that inhibits a NC_(Ca-ATP) channel or a pharmaceuticallyacceptable salt thereof. This pharmaceutical composition can beconsidered neuroprotective. For example, the pharmaceutical compositioncomprising a combination of the thrombolytic agent and a compound thatinhibits a NC_(Ca-ATP) channel is neuroprotective because it increasesthe therapeutic window of for the administration of the thrombolyticagent by several hours, for example the therapeutic window foradministration of thrombolytic agents may be increased by several hours(4-8 hrs) by co-administering antagonist of the NC_(Ca-ATP) channel.

The channel can be inhibited by an NC_(Ca-ATP) channel inhibitor, anNC_(Ca-ATP) channel blocker, a type 1 sulfonylurea receptor (SUR1)antagonist, SUR1 inhibitor, or a compound capable of reducing themagnitude of membrane current through the channel. More specifically,the SUR1 antagonist is selected from the group consisting ofglibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide,midaglizole, LY397364, LY389382, glyclazide, glimepiride, estrogen,estrogen related-compounds (estradiol, estrone, estriol, genistein,non-steroidal estrogen (e.g., diethystilbestrol), phytoestrogen (e.g.,coumestrol), zearalenone, etc.), and compounds known to inhibit or blockK_(ATP) channels. MgADP can also be used to inhibit the channel. Othercompounds that can be used to block or inhibit K_(ATP) channels include,but are not limited to tolbutamide, glyburide(1[p-2[5-chloro-O-anisamido)ethyl]phenyl]sulfonyl]-3-cyclohexyl-3-urea);chlopropamide (1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide(1-cyclohexyl-3[[p-[2(5-methylpyrazinecarboxamido)ethyl]phenyl]sulfonyl]urea); ortolazamide(benzenesulfonamide-N[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl).

Another embodiment of the present invention comprises a compositioncomprising a membrane preparation derived from a neural endothelial cellexpressing a NC_(Ca-ATP) channel, wherein channel is blocked byantagonists of type 1 sulfonylurea receptor (SUR1) and opened by SUR1activators. More specifically, the channel has the followingcharacteristics: (a) it is a 35 pS type channel; (b) it is stimulated bycytoplasmic Ca²⁺ in the concentration range from about 10⁻⁸ to about10⁻⁵ M; (c) it opens when cytoplasmic ATP is less than about 0.8 μM; and(d) it is permeable to the monovalent cations K⁺, Cs⁺, Li⁺ and Na⁺.

In further embodiments, the compound that inhibits the NC_(Ca-ATP)channel can be administered in combination with a thrombolytic agent(e.g., tissue plasminogen activator (tPA), urokinase, prourokinase,streptokinase, anistreplase, reteplase, tenecteplase), an anticoagulantor antiplatelet (e.g., aspirin, warfarin or coumadin), statins,diuretics, vasodilators (e.g., nitroglycerin), mannitol, diazoixde orsimilar compounds that stimulates or promotes ischemic precondition.

Still further, another embodiment comprises a method of treating anacute cerebral ischemia in a subject comprising administering to asubject an amount of a thrombolytic agent or a pharmaceuticallyacceptable salt thereof in combination with an amount of a compound thatinhibits a NC_(Ca-ATP) channel or a pharmaceutically acceptable saltthereof. In certain embodiments, the thrombolytic agent is a tissueplasminogen activator (tPA), urokinase, prourokinase, streptokinase,anistreplase, reteplase, tenecteplase or any combination thereof. TheSUR1 antagonist can be administered by any standard parenteral oralimentary route, for example the SUR1 antagonist may be administered asa bolus injection or as an infusion or a combination thereof.

The channel is expressed on neuronal cells, neuroglia cells, neuralepithelial cells or a combination thereof. The inhibitor blocks theinflux of Na⁺ into the cells thereby preventing depolarization of thecells. Inhibition of the influx of Na⁺ into the cells thereby preventscytotoxic edema and reduces hemorrhagic conversion. Thus, this treatmentreduces cell death or necrotic death of neuronal and/or neuralendothelial cells.

In certain embodiments, the amount of the SUR1 antagonist administeredto the subject is in the range of about 0.0001 μg/kg/day to about 20mg/kg/day, about 0.01 μg/kg/day to about 100 μg/kg/day, or about 100μg/kg/day to about 20 mg/kg/day. Still further, the SUR1 antagonist maybe administered to the subject in the from of a treatment in which thetreatment may comprise the amount of the SUR1 antagonist or the dose ofthe SUR1 antagonist that is administered per day (1, 2, 3, 4, etc.),week (1, 2, 3, 4, 5, etc.), month (1, 2, 3, 4, 5, etc.), etc. Treatmentsmay be administered such that the amount of SUR1 antagonist administeredto the subject is in the range of about 0.0001 μg/kg/treatment to about20 mg/kg/treatment, about 0.01 μg/kg/treatment to about 100μg/kg/treatment, or about 100 μg/kg/treatment to about 20mg/kg/treatment.

Another embodiment of the present invention comprises a method ofreducing mortality of a subject suffering from a stroke comprisingadministering to the subject a compound effective to inhibit aNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, a neuralendothelial cell or a combination thereof. The compound reduces strokesize and reduces edema located in the peri-infarct tissue. The compoundcan be administered alimentary (e.g., orally, buccally, rectally orsublingually) or parenterally (e.g., intravenously, intradermally,intramuscularly, intraarterially, intrathecally, subcutaneously,intraperitoneally, intraventricularly) and/or topically (e.g.,transdermally), mucosally, or by direct injection into the brainparenchyma.

Still further, another embodiment comprises a method of reducing edemain a peri-infarct tissue area of a subject comprising administering tothe subject a compound effective to inhibit a NC_(Ca-ATP) channel in aneuronal cell, a neuroglia cell, an endothelium cell or a combinationthereof.

Further embodiments comprises a method of treating a subject at risk fordeveloping a stroke comprising administering to the subject a compoundeffective to inhibit a NC_(Ca-ATP) channel in neuronal cell, a neurogliacell, a neural endothelial cell or a combination thereof.

In certain embodiments, the subject is undergoing treatment for acardiac condition, thus the condition increases the subjects risk fordeveloping a stroke. The treatment, for example, may comprise the use ofthrombolytic agents to treat myocardial infarctions. Still further, thesubject may be at risk for developing a stroke because the subjectsuffers from atrial fibrillation or a clotting disorder. Other subjectsthat are at risk for developing a stroke include subjects that are atrisk of developing pulmonary emboli, subjects undergoing surgery (e.g.,vascular surgery or neurological surgery), or subjects undergoingtreatments that increase their risk for developing a stroke, forexample, the treatment may comprise cerebral/endovascular treatment,angiography or stent placement.

Another embodiment of the present invention comprises a method oftreating a subject at risk for developing cerebral edema comprisingadministering to the subject a compound effective to inhibit aNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, a neuralendothelial cell or a combination thereof. The subject at risk may besuffering from an arterior-venous malformation, or a mass-occupyinglesion (e.g., hematoma) or may be involved in activities that have anincreased risk of brain trauma.

Yet further, another embodiment of the present invention comprises amethod of maintaining the integrity of the gliotic capsule surroundingbrain abscess of a subject comprising administering to the subject acompound effective to inhibit and/or block a NC_(Ca-ATP) channel in aneuronal cell, or a neuroglia cell, a neural endothelial cell or acombination thereof.

Still further, another method of the present invention comprises amethod of diagnosing neuronal cell edema and/or cytotoxic damage in thebrain comprising: labeling an antagonist of SUR1; administering thelabeled antagonist of SUR1 to a subject; measuring the levels of labeledantagonist of SUR1 in the brain of the subject, wherein the presence oflabeled antagonist of SUR1 indicates neuronal cell edema and/orcytotoxic damage in the brain.

Another method of the present invention comprise determining theboundaries of a brain tumor comprising: labeling an antagonist of SUR1;administering the labeled antagonist of SUR1 to a subject; visualizingthe labeled antagonist of SUR1 in the brain of the subject, wherein thepresence of labeled antagonist of SUR1 indicates the boundaries of thebrain tumor, for example, a metastatic tumor. In certain embodiments,the step of visualizing is performed using by using positron emissiontopography (PET) scans.

In further embodiments, the methods can comprise a method of determiningthe penumbra following a stroke comprising: labeling an antagonist ofSUR1; administering the labeled antagonist of SUR1 to a subject;visualizing the labeled antagonist of SUR1 in the brain of the subject,wherein the presence of labeled antagonist of SUR1 indicates thepenumbra.

Yet further, the present invention comprises a method monitoring strokeneural disease comprising: labeling an antagonist of SUR1; administeringthe labeled antagonist of SUR1 to a subject; visualizing the labeledantagonist of SUR1 in the brain of the subject, wherein the presence oflabeled antagonist of SUR1 indicates the progression of the disease. Incertain embodiments, the step is visualizing is performed daily tomonitor the progression of the stroke.

Another embodiment comprises a neuroprotective infusion kit comprising acompound that inhibits a NC_(Ca-ATP) channel in a neuronal cell, aneuroglia cell, a neural endothelial cell or a combination thereof andan IV solution. The compound and solution are contained within the samecontainer or within different containers. More specifically, thecompound is contained within the container of solution.

The kit may further comprise a neuroprotective bolus kit, wherein thebolus kit comprises a pre-loaded syringe of a compound inhibits aNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, a neuralendothelial cell or a combination thereof.

Still further, another embodiment comprises a neuroprotective kitcomprising a compound that inhibits NC_(Ca-ATP) channel in a neuronalcell, a neuroglia cell, an endothelium cell or a combination thereof anda thrombolytic agent (e.g., tPA), an anticoagulant (e.g., warfarin orcoumadin), an antiplatelet (e.g., aspirin), a diuretic (e.g., mannitol),a statin, or a vasodilator (e.g., nitroglycerin).

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1 shows that addition of exogenousphosphatidylinositol-4,5-bisphosphate (PIP₂) causes activation of theNC_(Ca-ATP) channel, despite the presence of ATP in the bath solution.Initially, channel activity was recorded in an inside-out patch ofmembrane from an R1 astrocyte, with a bath solution containing 1 μM Ca²⁺and 10 μM ATP, which was sufficient to block channel activity. Additionof 50 μM PIP₂ resulted in channel activation, reflecting an apparentdecrease in affinity of the channel for ATP.

FIG. 2 shows that the NC_(Ca-ATP) channel in an R1 astrocyte isinhibited by estrogen. The initial portion of the record shows briskactivity from a number of superimposed channels, recorded in a cellattached patch of membrane from an R1 astrocyte obtained from a female.Addition of 10 nM estrogen to the bath promptly resulted in stronginhibition of channel activity. The mechanism involved is believed to berelated to estrogen receptor mediated activation of phospholipase C(PLC), resulting in depletion of PIP₂ from the membrane, and reflectingan apparent increase in affinity for ATP.

FIGS. 3A-3B show Western blots demonstrating that R1 astrocytes fromboth males and females express estrogen receptors and SUR1, a marker ofthe NC_(Ca-ATP) channel. Cell lysates were obtained from gelatin spongeimplants from males (M) and females (F) and studied at two dilutions (4×and 1×), with lysates from uterus used as controls. FIG. 3A wasdeveloped using antibodies directed against estrogen receptors (ER),demonstrating that both ERα and ERβ are expressed in astrocytes fromboth genders. Western blots showed that SUR1 is also expressed by cellsfrom both genders, with pancreatic tissue used as control (FIG. 3B).

FIG. 4 shows that the NC_(Ca-ATP) channel in an R1 astrocyte from a maleis inhibited by estrogen. The initial portion of the record shows briskactivity from a number of superimposed channels, recorded in a cellattached patch of membrane from an R1 astrocyte obtained from a male.Addition of 10 nM estrogen to the bath promptly resulted in stronginhibition of channel activity.

FIGS. 5A-5D shows the gliotic capsule. FIG. 5A shows a coronal sectionof a rat brain sectioned though the site of implantation of a largegelatin sponge; the sponge (innermost dark region) is encapsulated by agliotic capsule (light area), outside of which is found a region ofvasogenic edema (outer dark area), identified by pre-mortemadministration of methylene blue. FIGS. 5B and 5C show low power andhigh power views, respectively, of the gliotic capsule immunolabeled forGFAP. FIG. 5D shows a high power view of GFAP-labeled cells inside ofthe gelatin sponge implant.

FIGS. 6A-6H show immunolabeled astrocytes. FIGS. 6A, 6C, 6E showfreshly-isolated large phase-bright R1 astrocytes immunolabeled for GFAP(FIG. 6C) and vimentin (FIG. 6E). FIGS. 6B,D,F show freshly-isolatedsmall phase-dark R2 astrocytes immunolabeled for GFAP (FIG. 6D) andvimentin (FIG. 6F). FIG. 6G shows primary cultures of astrocytesisolated from a gliotic capsule, with R1 astrocytes developing intolarge polygonal cells (FIG. 6Gb), and R2 astrocytes developing intosmall bipolar cells (FIG. 6Ga). FIG. 6H shows that R2 astrocytes, butnot R1 astrocytes, are labeled with fluorescein tagged chlorotoxinderived from the scorpion, Leiurus quinquestriatus.

FIGS. 7A-7D show that the inner zone of the gliotic capsule expressesSUR1 but not SUR2. Immunolabling for SUR1 (FIG. 7A) showed prominentexpression in cells adjacent to the gelatin sponge (gf), whereasimmunolabeling for SUR2 showed no expression (FIG. 7B). A single cellenzymatically isolated from a gelatin sponge implant and immunolabeledfor SUR1 is shown (FIG. 7C). FIG. 7D shown RT-PCR for SUR1 in controlinsulinoma cells (lane 2) and in isolated R1 astrocytes (lane 3), andfor SUR2 in control cardiac cells (lane 4), but not in isolated R1astrocytes (lane 5).

FIGS. 8A-8I show various features of the gliotic capsule. The glioticcapsule is characterized by GFAP-positive cells that are severalcell-layers thick (FIG. 8A). Only the inner zone of the gliotic capsuleis hypoxic, as demonstrated by pimonidozole labeling (FIG. 8B) and byimmunolabeling for HIF1α (FIG. 8C). Also, only the inner zone isimmunolabeled for SUR1 (FIG. 8D), and for the tight junction proteins,ZO-1 (FIG. 8E) and occludens (FIG. 8F). FIGS. 8G-8I show thatpimonidazole, HIF1α and occludens all localize to GFAP-positiveastrocytes that form the inner zone of the gliotic capsule.

FIGS. 9A-B show effects of NC_(Ca-ATP) channel inhibition (FIG. 9A) andNC_(Ca-ATP) channel activation (FIG. 9B) on the gliotic capsule. Animalswith gelatin sponge implants were treated with glibenclamide infusion(FIG. 9A) or diazoxide infusion (FIG. 9B) via osmotic mini-pumps thatdelivered the compounds directly into the area of the gelatin sponge.Immunolabeling for GFAP showed that channel inhibition withglibenclamide resulted in formation of a well defined gliotic capsule(FIG. 9A), whereas channel activation with diazoxide resulted information of a broader, ill-defined capsule (FIG. 9B), due todiazoxide-induced necrotic death of inner zone cells.

FIGS. 10A-B show that infusion of diazoxide into the area around thegelatin sponge resulted in a heavy infiltration of polymorphonuclearleukocytes (PMNs). Nuclear labeling with DAPI showed densely packedsmall cells in the vicinity of the gelatin sponge (FIG. 10A), withimmunolabeling using the PMN-specific marker, MMP-8, demonstrating thatthese cells were PMNs (FIG. 10B). It is believed that the stronginflammatory response represented by the infiltrating PMNs was due todisruption of the barrier between brain and foreign body (gelatinsponge) normally formed by the inner zone of the gliotic capsule.

FIGS. 11A-11L show that R1 astrocytes in the inner zone of the glioticcapsule typically express SUR1, a marker for the NC_(Ca-ATP) channel.The inner zones of the gliotic capsules in rats with gelatin spongeimplants (FIGS. 11A-11C), in rats with cerebral abscess (FIGS. 11D-11F),and in humans with metastatic tumor (FIGS. 11J-11L) are shown. Alsoshown is the area of reactive gloss adjacent to a stroke in the rat(FIGS. 11G-11I) resulting from occlusion of the middle cerebral artery.In all cases, a field of cells is labeled for GFAP and co-labelled forSUR1, as indicated. Examples of single cells at high power are alsoshown for each condition.

FIGS. 12A-12C shows that stellate astrocytes near the edge of a strokeup-regulate SUR1 (FIG. 12A), a marker of the NC_(Ca-ATP) channel. In themiddle of the stroke, cells with altered morphology including blebbingare also immunolabeled for SUR1 (FIGS. 12B,12C).

FIGS. 13A-13C show that glibenclamide protects from Na azide-inducedchannel opening and necrotic cell death. FIG. 13A shows phase contrastimages of 4 different freshly isolated R1 astrocytes observed over thecourse of 30 min each. The cell exposed to vehicle solution aloneremained phase bright with no pathological deterioration (control). Thecell depleted of ATP by exposure to Na azide (1 mM) developedprogressive blebbing consistent with cytotoxic edema. Similarly, thecell exposed to the NC_(Ca-ATP) channel opener, diazoxide, developedprogressive blebbing consistent with cytotoxic edema. The cell exposedto Na azide in the presence of glibenclamide remained phase bright withno pathological deterioration. FIGS. 13B and 13C show cell death ofisolated R1 astrocytes induced by ATP depletion in vitro. Freshlyisolated R1 astrocytes were labeled for necrotic death with propidiumiodide (PI) (FIG. 13B), or for apoptotic death with annexin V (FIG.13C), under control conditions, after exposure to Na azide (1 mM), orafter exposure to Na azide in the presence of glibenclamide (1 μM).Exposure to Na azide resulted mostly in necrotic death that was largelyprevented by glibenclamide.

FIGS. 14A-14L shows that SUR1 is up-regulated in MCA stroke. Watershedarea between MCA-ACA in 3 different animals 8-16 hr after MCA stroke,identified by pre-mortem administration of Evans blue and postmortemperfusion with India ink (FIG. 14A), by TTC staining (FIG. 14B) and byimmunofluorescence imaging for SUR1 (FIG. 14C). Immunofluorescenceimages showing SUR1 at 3 hr in the core of the stroke in cells (FIG.14D) double-labeled for the neuronal marker, NeuN (FIG. 14E), andshowing SUR1 at 8 hr in the peri-infarct region in cells (FIGS. 14G,14J) double-labeled for the astrocytic marker, GFAP (FIG. 14H), and theendothelial cell marker, von Willebrand factor (FIG. 14K). Superimposedimages of double-labeled fields are shown (FIGS. 14F, 14I, and 14L).

FIGS. 15A-15G show that SUR1 but not Kir6.1 or Kir6.2 istranscriptionally up-regulated in MCA stroke. Western blots for SUR1(≈180 kDa) at different times (FIG. 15A) and in different locations(FIG. 15B) after MCA stroke; in (FIG. 15A), lysates were all from TTC(+)peri-infarct regions of the involved hemisphere, obtained at the timesindicated; in (FIG. 15B), lysates were all obtained 8 hr after MCA stokefrom the regions indicated; each individual lane in a and b is from asingle animal. Quantification of the data from (FIG. 15A) and (FIG.15B), respectively, combined with comparable data for Kir6.1 and Kir6.2;for each individual blot, data were normalized to values of β-actin andto the control data for that blot and analyzed separately; **, p<0.01.In situ hybridization for SUR1, 3 hr after MCA stroke; paraffin sectionsshowed that large neuron-like cells (FIG. 15E) and capillaries (FIG.15F) in the ischemic zone were labeled, whereas tissues from the sameareas on the control side were not (FIG. 15G).

FIGS. 16A-16D show patch clamp recordings of NC_(Ca-ATP) channel inneuron-like cells in stroke. FIG. 16A shows phase-contrast image oflarge neuron-like cells enzymatically isolated from ischemic region 3 hrfollowing MCAO. FIG. 16B shows recording of inside-out patch using Cs⁺as the charge carrier; channel activity was blocked by glibenclamidegiven as indicated (arrow); a and b show expanded records of theportions indicated. FIG. 16C shows recordings at potentials indicated ofinside-out patch using K⁺ as the charge carrier; channel activity wasblocked by glibenclamide. FIG. 16D shows a plot of single channelamplitudes at different voltages showing single channel slopeconductance of 34 pS.

FIGS. 17A-17E show that glibenclamide reduces mortality, edema andstroke size in MCA stroke. In FIG. 17A, Mortality was assessed during 7days after MCA stroke [double occlusion model with malignant cerebraledema (MCE)] in two treatment groups, each comprised of 19 female and 10male rats, treated with either saline (empty symbols) or glibenclamide(filled symbols); mortality at 7 days was significantly different.Subgroup analyses for males and females showed similar results. In FIG.17B edema was assessed 8 hr after MCA stroke (MCE model) in twotreatment groups, each comprised of 6 male rats treated with eithersaline or glibenclamide; tissues were first processed with TTC to allowseparation into TTC(+) and TTC(−) portions of the involved hemisphereand contralateral hemisphere, prior to determining wet/dry weights;values in TTC(+) regions were statistically different. In FIGS. 17C-17E,stroke size was assessed 48 hr after MCA stroke [thromboembolic (TE)model] in two treatment groups, each comprised of 10 male rats, treatedwith either saline or glibenclamide; images of TTC-stained coronalsections following MCA stroke (TE model) in an animal treated withsaline (FIG. 17C) and another treated with glibenclamide (FIG. 17D),showing cortical sparing often associated with glibenclamide treatment;values of stroke size, expressed as percent of hemisphere volume (FIG.17E).

FIGS. 18A-18D show that tissue distribution of BODIPY-glibenclamide inMCA stroke. a-c, Fluorescence images of brain sections in an animal 6 hrafter MCA stroke (MCE model) and administration of BODIPY-glibenclamide;fluorescent labeling was evident in cells, microvessels (FIG. 18A) andcapillaries (FIG. 18C) from ischemic regions, but not in thecontralateral hemisphere (FIG. 18B); the images in (FIGS. 18A, 18B) arefrom the same animal, taken with the same exposure time; in (FIG. 18C),the single layer of nuclei confirms that the structure brightly labeledby BODIPY-glibenclamide is a capillary. In FIG. 18D, immunofluorescenceimage of a brain section from an animal 6 hr after MCA stroke (MCEmodel) labeled with anti-SUR1 antibody showing strong labeling in acapillary and in adjacent neuron-like cells.

FIGS. 19A-9H show that glibenclamide reduces hemorrhagic conversion.FIGS. 19A-19D are from animals co-treated with saline; FIGS. 19E-19H arefrom animals co-treated with glibenclamide The left column ofphotographs of coronal sections shows, in rows 1-2 only,intraventricular hemorrhage, plus large areas of hemorrhagic conversionin ischemic cortical/subcortical regions (red areas on the right side ofpictures; arrows). The right column of photographs of TTC-processedsections from the same animals show the areas of infarction.

FIGS. 20A-20B show zymography showing gelatinase activity of matrixmetalloproteinases (MMP's) in stroke, and absence of direct MMPinhibition by glibenclamide. FIG. 20A shows activation of MMP-9 & MMP-2in stroke tissue compared to control; activity of recombinant MMP-9 &MMP-2 shown at left. FIG. 20B shows gelatinase activity of recombinantenzyme and stroke tissue under control conditions (CTR), in presence ofglibenclamide (10 μM), and in presence of MMP inhibitor II (300 nM;Calbiochem).

FIG. 21 shows phase contrast photomicrograph of cerebral capillariesfreshly isolated from normal brain, after enzymatic cleaning inpreparation for patch clamping.

FIGS. 22A-22F show that freshly isolated cerebral endothelial and smoothmuscle cells are readily distinguished electrophysiologically. FIGS. 22Aand 22B show superimposed macroscopic currents recorded during 200 msdepolarizing pulses from −120 mV to +120 mV in 20 mV steps in anendothelial cell (FIG. 22A) and in an elongated smooth muscle cell (FIG.22B); holding potential, −60 mV; nystatin perforated patch technique;bath solution, standard Krebs with 2 mM Ca²⁺; pipette solution, 145 mMK⁺. FIGS. 22C and 22D show current-voltage curves computed from average(mean±SE) currents at the end of 200-ms test pulses recorded in 9endothelial cells (FIG. 22C) and 7 smooth muscle cells (FIG. 22D); sameholding potential, technique and solutions as in FIGS. 22A and 22B.FIGS. 22E and 22F show current voltage curves recorded during ramppulses (0.45 mV/ms, holding potential, −60 mV) in an endothelial cell(FIG. 22E) and in a smooth muscle cell (FIG. 22F); same holdingpotential, technique and bath solution as in FIGS. 22A and 22B, but withpipette solution containing 145 mM Cs⁺ instead of K⁺.

FIG. 23 shows real time RT-PCR showing up-regulation of SUR1-mRNA instroke.

FIGS. 24A-24E show SUR1 knock down (SUR1KD) in R1 astrocytes protectsfrom ATP-depletion-induced depolarization. FIGS. 24A and 24B showWestern blot (FIG. 24A) and quantification of Western blots (FIG. 24B)of R1 cell lysates confirmed knock down of SUR1 expression by antisense.FIGS. 24C-24E show Na azide caused large depolarizations in cellsexposed to SCR-ODN (FIGS. 24C, 24E) but little or no depolarization incells exposed to AS-ODN (FIGS. 24D, 24E).

FIGS. 25A-25F show transcription factors in stroke. Immunofluorescenceimages of subcortical watershed region between ACA and MCA territories,from ipsilateral peri-infarct tissues 8 hr after MCAO (FIGS. 25A-D) andfrom contralateral control tissues (FIGS. 25E, 25F). The peri-infarctregion showed up-regulation of both transcription factors, Sp1 (FIGS.25A, 25C) and HIF1α (FIG. 25B) in neuron-like cells and capillaries, aswell as SUR1 in capillaries (FIG. 25D). Control tissues showed littleSp1 and no HIF1α (FIGS. 25E and 25F).

FIGS. 26A-26C show an increase in nuclear localization of thetranscription factor, SP1, and SP1 co-localization with SUR1 in strokeImmunofluorescence images showing increase of nuclear SP1 labeling inischemic area 3-hr after MCAO (FIG. 26B), compared to contralateral side(FIG. 26A). FIG. 26C double labeling of large neuron-like cell showingnuclear SP1 (green) and cytoplasmic/plasmalemmal SUR1 (red) in the samecell.

FIGS. 27A-27 show regulation of SUR1 expression by the transcriptionfactor, HIF1α. FIGS. 27A and 27C show Western blot analysis of HIF1αprotein in R1 astrocytes from gelfoam implant model of control (CTR) andHIF1α knock-down (KD). FIGS. 27B and 27C show SUR1 protein in the samecell lysates.

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

I. DEFINITIONS

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternative are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.”

As used herein, the term “acute” refers to the onset of a health effect,usually the effect is a rapid onset that is considered brief, notprolonged.

As used herein, the term “acute cerebral ischemia” refers to a cerebralischemic event that has a rapid onset and is not prolonged. The terms“acute cerebral ischemia” and “stroke” can be used interchangeably.”

As used herein, the term “anti-cancer therapy” or “anti-tumor” refers toany therapy that destroys a cancer cell and/or a tumor cell, or slows,arrests, or reverses the growth of a cancer cell and/or tumor cell.Anti-cancer or anti-tumor therapies include, without limitation,radiation therapy (radiotherapy), chemotherapy, or a combination ofthese therapies.

As used herein, the term “agonist” refers to a biological or chemicalagent that combines with a receptor on a cell and initiates the same orequivalent reaction or activity produced by the binding of an endogenoussubstance. In the present invention, the agonist combines, binds, and/orassociates with a NC_(Ca-ATP) channel of a neuronal cell, a neuroglialcell, or a neural endothelial cell, such that the NC_(Ca-ATP) channel isopened (activated). In certain embodiments, the agonist combines, bindsand/or associates with a regulatory subunit of the NC_(Ca-ATP) channel,particularly a SUR1. Alternatively, the agonist combines, binds, and/orassociates with a pore-forming subunit of the NC_(Ca-ATP) channel, suchthat the NC_(Ca-ATP) channel is opened (activated). The terms agonistand/or activator can be used interchangeably.

As used herein, the term “antagonist” refers to a biological or chemicalagent that acts within the body to reduce the physiological activity ofanother chemical or biological substance. In the present invention, theantagonist blocks, inhibits, reduces and/or decreases the activity of aNC_(Ca-ATP) channel of a neuronal cell, a neuroglia cell or a neuralendothelial cell (e.g., capillary endothelial cells). In the presentinvention, the antagonist combines, binds, associates with a NC_(Ca-ATP)channel of neuronal cell, a neuroglia cell or a neural endothelial cell(e.g., capillary endothelial cells), such that the NC_(Ca-ATP) channelis closed (deactivated), meaning reduced biological activity withrespect to the biological activity in the diseased state. In certainembodiments, the antagonist combines, binds and/or associates with aregulatory subunit of the NC_(Ca-ATP) channel, particularly a SUR1.Alternatively, the antagonist combines, binds, and/or associates with apore-forming subunit of the NC_(Ca-ATP) channel, such that theNC_(Ca-ATP) channel is closed (deactivated). The terms antagonist orinhibitor can be used interchangeably.

As used herein, the terms “brain abscess” or “cerebral abscess” refer toa circumscribed collection of purulent exudate that is typicallyassociated with swelling.

As used herein, the terms “blood brain barrier” or “BBB” refer thebarrier between brain blood vessels and brain tissues whose effect is torestrict what may pass from the blood into the brain.

As used herein, the term “cancer” refers to a hyperproliferation ofcells whose unique trait—loss of normal controls—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Cancer may include a tumor comprised of tumor cells. Those of skill inthe art understand that not all cancers comprise tumor cells, forexample leukemia does not comprise tumor cells.

As used herein, the term “cerebral ischemia” refers to a lack ofadequate blood flow to an area, for example a lack of adequate bloodflow to the brain, which may be the result of a blood clot, blood vesselconstriction, a hemorrhage or tissue compression from an expanding mass.

As used herein, the term “depolarization” refers to an increase in thepermeability of the cell membrane to sodium ions wherein the electricalpotential difference across the cell membrane is reduced or eliminated.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” are interchangeable and refer to an amount thatresults in an improvement or remediation of the symptoms of the diseaseor condition. Those of skill in the art understand that the effectiveamount may improve the patient's or subject's condition, but may not bea complete cure of the disease and/or condition.

As used herein, the term “endothelium” refers a layer of cells that linethe inside surfaces of body cavities, blood vessels, and lymph vesselsor that form capillaries.

As used herein, the term “endothelial cell” refers to a cell of theendothelium or a cell that lines the surfaces of body cavities, forexample, blood or lymph vessels or capillaries. In certain embodiments,the term endothelial cell refers to a neural endothelial cell or anendothelial cell that is part of the nervous system, for example thecentral nervous system or the brain.

As used herein, the term “hyperproliferative disease” refers to adisease that results from a hyperproliferation of cells. Exemplaryhyperproliferative diseases include, but are not limited to cancer,tumors or neoplasms.

As used herein, the term “gliotic capsule” refers to a physical barriersurrounding, in whole or in part, a foreign body, including a metastatictumor, a cerebral abscess or other mass not normally found in brainexcept under pathological conditions. In certain embodiments, thegliotic capsule comprises an inner zone comprising neuronal cells,neuroglial cells (e.g., astrocytes) and/or endothelial cells expressinga NC_(Ca-ATP) channel.

The term “morbidity” as used herein is the state of being diseased. Yetfurther, morbidity can also refer to the disease rate or the ratio ofsick subjects or cases of disease in to a given population.

The term “mortality” as used herein is the state of being mortal orcausing death. Yet further, mortality can also refer to the death rateor the ratio of number of deaths to a given population.

As used herein, the term “neuronal cell” refers to a cell that is amorphologic and functional unit of the nervous system. The cellcomprises a nerve cell body, the dendrites, and the axon. The termsneuron, nerve cell, neuronal, neurone, and neurocyte can be usedinterchangeably. Neuronal cell types can include, but are not limited toa typical nerve cell body showing internal structure, a horizontal cell(of Cajal) from cerebral cortex; Martinottic cell, biopolar cell,unipolar cell, Pukinje cell, and a pyramidal cell of motor area ofcerebral cortex.

As used herein, the term “neural” refers to anything associated with thenervous system.

As used herein, the terms “neuroglia” or “neuroglial cell” refers to acell that is a non-neuronal cellular element of the nervous system. Theterms neuroglia, neurogliacyte, and neuroglial cell can be usedinterchangeably. Neuroglial cells can include, but are not limited toependymal cells, astrocytes, oligodendrocytes, or microglia.

The term “preventing” as used herein refers to minimizing, reducing orsuppressing the risk of developing a disease state or parametersrelating to the disease state or progression or other abnormal ordeleterious conditions.

As used herein, the term “stroke” refers to any acute, clinical eventrelated to the impairment of cerebral circulation. The terms “acutecerebral ischemia” and “stroke” can be used interchangeably.”

As used herein, the term “tumor” refers to any swelling tumefaction.Tumor is interchangeable with the term “neoplasm” which is abnormaltissue growth. Tumors can be malignant or benign.

As used herein, the term “tumor-brain barrier” refers to a biochemicalbarrier between a foreign body in the brain and the surrounding tissueof the brain. The tumor-brain barrier is interchangeably referred toherein as TBB.

The terms “treating” and “treatment” as used herein refer toadministering to a subject a therapeutically effective amount of acomposition so that the subject has an improvement in the disease orcondition. The improvement is any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient's condition, but may not be a complete cure of the disease.Treating may also comprise treating subjects at risk of developing adisease and/or condition.

II. THE PRESENT INVENTION

The present invention is directed to therapeutic compositions andmethods of using the same. In one embodiment, the therapeuticcomposition is an agonist and/or antagonist of a NC_(Ca-ATP) channel ofa neuronal cell, a neuroglial cell, or a neural endothelial cell.

In certain embodiments, the present invention is directed to a method oftreating a cancer patient in need of such treatment comprisingadministering an agonist of a NC_(Ca-ATP) channel of an astrocyte,wherein the agonist activates a NC_(Ca-ATP) channel. In specificembodiments, the agonist targets a SUR1 of the NC_(Ca-ATP) channel. Incertain embodiments, the cancer is located in the brain and, morespecifically, comprises a metastatic tumor located in the brain.

In certain embodiments, the agonist of the present invention disruptsthe integrity of the tumor-brain barrier surrounding the cancer, therebypermitting access to otherwise barred agents across the tumor-brainbarrier. In certain embodiments, the agonist is administered incombination with an anti-cancer therapy, including chemotherapy,radiotherapy and/or immunotherapy.

Alternatively, the present invention is directed to a method ofdisrupting a tumor-brain barrier comprising administering an agonist ofa NC_(Ca-ATP) channel of an astrocyte, wherein said agonist activatessaid NC_(Ca-ATP) channel.

Methods involving an agonist of the NC_(Ca-ATP) channel are directed toselectively killing neuronal cells, neuroglial cells (e.g., astrocytes)and/or neural endothelial cells expressing the NC_(Ca-ATP) channel byinfusion of an agonist of the NC_(Ca-ATP) channel, such as diazoxide, tothe astrocyte. The infusion can be direct or indirect. Selective killingof neuronal cells, neuroglial cells (e.g., astrocytes) and/or neuralendothelial cells are desirable when treating a pathology involving agliotic capsule, such as a metastatic brain tumor. The agonistfacilitates mounting an immune response, or, alternatively, improvespermeability for chemotherapeutic agents.

As described herein, the sulfonylurea receptor 1 (SUR1) is expressed inR1 astrocytes as part of the NC_(Ca-ATP) channel, which make up thetumor-brain barrier (TBB) in brain metastasis. Targeting the SUR1 of theR1 astrocytes with an agonist thereof compromises the integrity of theTBB, thereby providing a treatment mechanism for metastatic tumors inthe brain. In specific embodiments, the agonists of the presentinvention disrupt the integrity of the gliotic capsule surrounding theforeign body, thereby permitting entry of otherwise barred biologicaland/or endogenous compounds, such as leukocytes, into the glioticcapsule.

In certain embodiments, the agonists include, for example, a compoundcapable of opening, activating and/or increasing the activity of anneuronal cells, neuroglial cells (e.g., astrocytes) and/or neuralendothelial cells expressing NC_(Ca-ATP) channel. Specifically, theagonists are SUR1 activators such as, diazoxide and the like, which areknown in the art to open (activate) K channels.

The present invention is contemplated for use in combination withchemotherapy, immunotherapy and/or radiotherapy. In the treatment ofsolid tumors (e.g., tumors in the lung, colon, breast, and brain),efficient treatment is hindered by the difficulty in penetrating thetumor mass with anti-cancer agents (Jain, 1994). The identification of ameans by which to facilitate the delivery of therapeutic agents to thecancer site is needed to enhance the effectiveness of currentanti-cancer therapies. To address this need, in alternative embodiments,Applicants provide herein methods for enhancing, improving and/orincreasing anti-cancer therapies by administering an antagonist of aNC_(Ca-ATP) channel.

For in vitro work, various solid tumor models may be used, such as, forexample, the well-recognized inducible breast cancer model, from whichtumor cells may be harvested and re-implanted into the brain to produceautologous “metastatic” tumors.

In addition to the sulfonylurea receptor 1 (SUR1) being expressed in R1astrocytes as part of the NC_(Ca-ATP) channel, the present inventionfurther describes that the SUR1 regulatory subunit of this channel isup-regulated in neurons and capillary endothelial cells followingischemia, and blocking this receptor reduces stroke size, cerebral edemaand mortality. Thus, antagonists of the NC_(Ca-ATP) channel may have animportant role in preventing, alleviating, inhibiting and/or abrogatingthe formation of cytotoxic and ionic edema.

In other embodiments, the therapeutic compound of the present inventioncomprises an antagonist of a NC_(Ca-ATP) channel of a neuronal cell, aneuroglial cell, a neural endothelial cell or a combination thereof.Antagonists are contemplated for use in treating adverse conditionsassociated with hypoxia and/or ischemia that result in increasedintracranial pressure and/or cytotoxic edema of the central nervoussystem. Such conditions include trauma, ischemic brain injury, namelysecondary neuronal injury, and hemorrhagic infarction. Antagonistsprotect the cells expressing the NC_(Ca-ATP) channel, which is desirablefor clinical treatment in which gliotic capsule integrity is importantand must be maintained to prevent the spread of infection, such as witha brain abscess. The protection via inhibition of the NC_(Ca-ATP)channel is associated with a reduction in cerebral edema.

In one aspect, the NC_(Ca-ATP) channel is blocked, inhibited, orotherwise is decreased in activity. In such examples, an antagonist ofthe NC_(Ca-ATP) channel is administered and/or applied. The antagonistmodulates the NC_(Ca-ATP) channel such that flux through the channel isreduced, ceased, decreased and/or stopped. The antagonist may have areversible or an irreversible activity with respect to the activity ofthe NC_(Ca-ATP) channel of the neuronal cell, neuroglial cell,endothelial cell or a combination thereof. The antagonist may prevent orlessen the depolarization of the cells thereby lessening cell swellingdue to osmotic changes that can result from depolarization of the cells.Thus, inhibition of the NC_(Ca-ATP) channel can reduce cytotoxic edemaand death of endothelial cells.

Subjects that can be treated with the therapeutic composition of thepresent invention include, but are not limited subjects suffering fromor at risk of developing conditions associated hypoxia and/or ischemiathat result in increased intracranial pressure and/or with cytotoxicedema of the central nervous system (CNS). Such conditions include, butare not limited to trauma (e.g., traumatic brain injury (TBI),concussion) ischemic brain injury, hemorrhagic infarction, stroke,atrial fibrillations, clotting disorders, pulmonary emboli,arterio-venous malformations, mass-occupying lesions (e.g., hematomas),etc. Still further subjects at risk of developing such conditions caninclude subjects undergoing treatments that increase the risk of stroke,for example, surgery (vascular or neurological), treatment of myocardialinfarction with thrombolytics, cerebral/endovascular treatments, stentplacements, angiography, etc.

Another aspect of the present invention comprises co-administration ofan antagonist of the NC_(Ca-ATP) channel with a thrombolytic agent.Co-administration of these two compounds increase the therapeutic windowof the thrombolytic agent by reducing hemorrhagic conversion. Thetherapeutic window for thrombolytic agents may be increased by several(4-8) hours by co-administering antagonist of the NC_(Ca-ATP) channel.

In addition to a thrombolytic agent, other agents can be used incombination with the antagonist of the present invention, for example,but not limited to antiplatelets, anticoagulants, vasodilators, statins,diuretics, etc.

Another aspect of the present invention comprises the use of labeledSUR1 antagonists to diagnose, determine or monitor stages of stroke,cerebral edema or visualize the size/boundaries/borders of a tumorand/or the stroke. For example, the penumbra following the stroke may bemonitored or visualized using labeled SUR1 antagonists.

Yet further, the compositions of the present invention can be used toproduce neuroprotective kits that are used to treat subjects at risk orsuffering from conditions that are associated with cytotoxic cerebraledema.

III. NC_(CA-ATP) CHANNEL

The invention is based, in part, on the discovery of a specific channel,the NC_(Ca-ATP) channel, defined as a channel on astrocytes in USApplication Publication No. 20030215889, which is incorporated herein byreference in its entirety. More specifically, the present invention hasfurther defined that this channel is not only expressed on astrocytes,it is expressed on neural cells, neuroglial cells, and/or neuralendothelial cells after brain trauma, for example, an hypoxic event, anischemic event, or other secondary neuronal injuries relating to theseevents.

The NC_(Ca-ATP) channel is activated by calcium ions (Ca²⁺) and issensitive to ATP. Thus, this channel is a non-selective cation channelactivated by intracellular Ca²⁺ and blocked by intracellular ATP. Whenopened by depletion of intracellular ATP, this channel is responsiblefor complete depolarization due to massive Na⁺ influx, which creates anelectrical gradient for Cl⁻ and an osmotic gradient for H₂O, resultingin cytotoxic edema and cell death. When the channel is blocked orinhibited, massive Na⁺ does not occur thereby preventing cytotoxicedema.

Certain functional characteristics distinguishes the NC_(Ca-ATP) channelfrom other known ion channels. These characteristics can include, butare not limited to 1) it is a non-selective cation channels that readilyallows passage of Na⁺, K⁺ and other monovalent cations; 2) it isactivated by an increase in intracellular calcium, and/or by a decreasein intracellular ATP; 3) it is regulated by sulfonylurea receptor type 1(SUR1), which heretofore had been considered to be associatedexclusively with K_(ATP) channels such as those found in pancreatic βcells.

More specifically, the NC_(Ca-ATP) channel of the present invention hasa single-channel conductance to potassium ion (K⁺) between 20 and 50 pS.The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on the cytoplasmicside of the cell membrane in a physiological concentration range, whereconcentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channel isalso inhibited by cytoplasmic ATP in a physiological concentrationrange, where the concentration range is from 10⁻¹ to 10 M. TheNC_(Ca-ATP) channel is also permeable to the following cations; K⁺, Cs⁺,Li⁺, Na⁺; to the extent that the permeability ratio between any two ofthe cations is greater than 0.5 and less than 2.

IV. MODULATORS OF THE NC_(CA-ATP) CHANNEL

The present invention comprises modulators of the channel, for exampleagonists and/or antagonist of the channel. Examples of antagonist oragonist of the present invention may encompass agonist and/orantagonists identified in US Application Publication No. 20030215889,which is incorporated herein by reference in its entirety. One of skillin the art is aware that the NC_(Ca-ATP) channel is comprised to twosubunits, the regulatory subunit, SUR1, and the pore forming subunit.

A. Modulators of SUR1

In certain embodiments, antagonists to sulfonylurea receptor-1 (SUR1)are suitable for blocking the channel. Examples of suitable SUR1antagonists include, but are not limited to glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, estrogen related-compounds estrogenrelated-compounds (estradiol, estrone, estriol, genistein, non-steroidalestrogen (e.g., diethystilbestrol), phytoestrogen (e.g., coumestrol),zearalenone, etc.) and combinations thereof. In a preferred embodimentof the invention the SUR1 antagonists is selected from the groupconsisting of glibenclamide and tolbutamide. Yet further, anotherantagonist can be MgADP. Other antagonist include blockers of K_(ATP)channels, for example, but not limited to tolbutamide, glyburide (1[p-2[5-chloro-O-anisamido)ethyl]phenyl]sulfonyl]-3-cyclohexyl-3-urea);chlopropamide (1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide(1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido)ethyl]phenyl]sulfonyl]urea); ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl).

Agonists that can be used in the present invention include, but are notlimited to agonist of SUR1, for example, diazoxide, pinacidil, P1075,cromakalin or activators of K_(ATP) channels. Other agonists caninclude, but are not limited to diazoixde derivatives, for example3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC55-9216), 6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine1,1-dioxide (BPDZ 154),7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ73), 6-Chloro-3-isopropylamino-4 H-thieno[3,2-e]-1,2,4-thiadiazine1,1-dioxide (NNC 55-0118)4, 6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414),3-(3-methyl-2-butylamino)-4H-pyrido [4,3-e]-1,2,4-thiadiazine1,1-dioxide (BPDZ 44),3-(1′,2′,2′-trimethylpropyl)amino-4H-pyrido(4,3-e)-1,2,4-thiadiazine1,1-dioxide (BPDZ 62), 3-(1′,2′,2′-trimethylpropyl)amine-4H-pyrido(2,3-e)-1,2,4-thiadiazine, 1,1-dioxide (BPDZ 79),2-alkyl-3-alkylamino-2H-benzo- and2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides,6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxidederivatives, 4-N-Substituted and -unsubstituted 3-alkyl- and3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides. Inaddition, other compounds, including 6-chloro-2-methylquinolin-4(1H)-one(HEI 713) and LN 533021, as well as the class of drugs,arylcyanoguanidines, are known activators or agonist of SUR1.

B. Modulators of SUR1 Transcription and/or Translation

In certain embodiments, the modulator can be a compound (protein,nucleic acid, siRNA, etc.) that modulates transcription and/ortranslation of SUR1 (regulatory subunit) and/or the molecular entitiesthat comprise the pore-forming subunit.

1. Transcription Factors

Transcription factors are regulatory proteins that binds to a specificDNA sequence (e.g., promoters and enhancers) and regulate transcriptionof an encoding DNA region. Thus, transcription factors can be used tomodulate the expression of SUR1. Typically, a transcription factorcomprises a binding domain that binds to DNA (a DNA binding domain) anda regulatory domain that controls transcription. Where a regulatorydomain activates transcription, that regulatory domain is designated anactivation domain. Where that regulatory domain inhibits transcription,that regulatory domain is designated a repression domain. Morespecifically, transcription factors such as Sp1 and HIF1α can be used tomodulate expression of SUR1.

2. Antisense and Ribozymes

An antisense molecule that binds to a translational or transcriptionalstart site, or splice junctions, are ideal inhibitors. Antisense,ribozyme, and double-stranded RNA molecules target a particular sequenceto achieve a reduction or elimination of a particular polypeptide, suchas SUR1. Thus, it is contemplated that antisense, ribozyme, anddouble-stranded RNA, and RNA interference molecules are constructed andused to modulate SUR1.

a) Antisense Molecules

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with complementary sequences. By complementary, it is meantthat polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNAs, are employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject.

Antisense constructs are designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructsmay include regions complementary to intron/exon splice junctions. Thus,antisense constructs with complementarity to regions within 50-200 basesof an intron-exon splice junction are used. It has been observed thatsome exon sequences can be included in the construct without seriouslyaffecting the target selectivity thereof. The amount of exonic materialincluded will vary depending on the particular exon and intron sequencesused. One can readily test whether too much exon DNA is included simplyby testing the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

It is advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

b) RNA Interference

It is also contemplated in the present invention that double-strandedRNA is used as an interference molecule, e.g., RNA interference (RNAi).RNA interference is used to “knock down” or inhibit a particular gene ofinterest by simply injecting, bathing or feeding to the organism ofinterest the double-stranded RNA molecule. This technique selectively“knock downs” gene function without requiring transfection orrecombinant techniques (Giet, 2001; Hammond, 2001; Stein P, et al.,2002; Svoboda P, et al., 2001; Svoboda P, et al., 2000).

Another type of RNAi is often referred to as small interfering RNA(siRNA), which may also be utilized to inhibit SUR1. A siRNA maycomprises a double stranded structure or a single stranded structure,the sequence of which is “substantially identical” to at least a portionof the target gene (See WO 04/046320, which is incorporated herein byreference in its entirety). “Identity,” as known in the art, is therelationship between two or more polynucleotide (or polypeptide)sequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match of the order ofnucleotides between such sequences. Identity can be readily calculated.See, for example: Computational Molecular Biology, Lesk, A. M., ed.Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ea., Academic Press, New York, 1993, andthe methods disclosed in WO 99/32619, WO 01/68836, WO 00/44914, and WO01/36646, specifically incorporated herein by reference. While a numberof methods exist for measuring identity between two nucleotidesequences, the term is well known in the art. Methods for determiningidentity are typically designed to produce the greatest degree ofmatching of nucleotide sequence and are also typically embodied incomputer programs. Such programs are readily available to those in therelevant art. For example, the GCG program package (Devereux et al.),BLASTP, BLASTN, and FASTA (Atschul et al.,) and CLUSTAL (Higgins et al.,1992; Thompson, et al., 1994).

Thus, siRNA contains a nucleotide sequence that is essentially identicalto at least a portion of the target gene, for example, SUR1, or anyother molecular entity associated with the NC_(Ca-ATP) channel such asthe pore-forming subunit. One of skill in the art is aware that thenucleic acid sequences for SUR1 are readily available in GenBank, forexample, GenBank accession L40624, which is incorporated herein byreference in its entirety. Preferably, the siRNA contains a nucleotidesequence that is completely identical to at least a portion of thetarget gene. Of course, when comparing an RNA sequence to a DNAsequence, an “identical” RNA sequence will contain ribonucleotides wherethe DNA sequence contains deoxyribonucleotides, and further that the RNAsequence will typically contain a uracil at positions where the DNAsequence contains thymidine.

One of skill in the art will appreciate that two polynucleotides ofdifferent lengths may be compared over the entire length of the longerfragment. Alternatively, small regions may be compared. Normallysequences of the same length are compared for a final estimation oftheir utility in the practice of the present invention. It is preferredthat there be 100% sequence identity between the dsRNA for use as siRNAand at least 15 contiguous nucleotides of the target gene (e.g., SUR1),although a dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater mayalso be used in the present invention. A siRNA that is essentiallyidentical to a least a portion of the target gene may also be a dsRNAwherein one of the two complementary strands (or, in the case of aself-complementary RNA, one of the two self-complementary portions) iseither identical to the sequence of that portion or the target gene orcontains one or more insertions, deletions or single point mutationsrelative to the nucleotide sequence of that portion of the target gene.siRNA technology thus has the property of being able to toleratesequence variations that might be expected to result from geneticmutation, strain polymorphism, or evolutionary divergence.

There are several methods for preparing siRNA, such as chemicalsynthesis, in vitro transcription, siRNA expression vectors, and PCRexpression cassettes. Irrespective of which method one uses, the firststep in designing an siRNA molecule is to choose the siRNA target site,which can be any site in the target gene. In certain embodiments, one ofskill in the art may manually select the target selecting region of thegene, which may be an ORF (open reading frame) as the target selectingregion and may preferably be 50-100 nucleotides downstream of the “ATG”start codon. However, there are several readily available programsavailable to assist with the design of siRNA molecules, for examplesiRNA Target Designer by Promega, siRNA Target Finder by GenScriptCorp., siRNA Retriever Program by Imgenex Corp., EMBOSS siRNA algorithm,siRNA program by Qiagen, Ambion siRNA predictor, Ambion siRNA predictor,Whitehead siRNA prediction, and Sfold. Thus, it is envisioned that anyof the above programs may be utilized to produce siRNA molecules thatcan be used in the present invention.

c) Ribozymes

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Forster and Symons,1987). For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., 1981; Reinhold-Hurek and Shub, 1992). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequencespecific cleavage/ligation reactions involving nucleic acids (Joyce,1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 reportsthat certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression is particularly suitedto therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990;Sioud et al., 1992). Most of this work involved the modification of atarget mRNA, based on a specific mutant codon that is cleaved by aspecific ribozyme. In light of the information included herein and theknowledge of one of ordinary skill in the art, the preparation and useof additional ribozymes that are specifically targeted to a given genewill now be straightforward.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992; Yuan and Altman, 1994), hairpinribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993)and hepatitis δ virus based ribozymes (Perrotta and Been, 1992). Thegeneral design and optimization of ribozyme directed RNA cleavageactivity has been discussed in detail (Haseloff and Gerlach, 1988;Symons, 1992; Chowrira, et al., 1994; and Thompson, et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozymes,the cleavage site is a dinucleotide sequence on the target RNA, uracil(U) followed by either an adenine, cytosine or uracil (A,C or U;Perriman, et al., 1992; Thompson, et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al. (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in SUR1 targeted ribozymes is simply a matter of preparing andtesting a given sequence, and is a routinely practiced screening methodknown to those of skill in the art.

C. Methods of Screening for Modulators

Further embodiments of the present invention can include methods foridentifying modulators of the NC_(Ca-ATP) channel, for example, agonistor antagonist, that modify the activity and/or expression. These assaysmay comprise random screening of large libraries of candidatesubstances; alternatively, the assays may be used to focus on particularclasses of compounds selected with an eye towards structural attributesthat are believed to make them more likely to modulate the function oractivity or expression of the NC_(Ca-ATP) channel.

By function, it is meant that one may assay for mRNA expression, proteinexpression, protein activity, or channel activity, more specifically,the ability of the modulator to open or inhibit or block the NC_(Ca-ATP)channel. Thus, the compounds for screening in accordance with theinvention include, but are not limited to natural or synthetic organiccompounds, peptides, antibodies and fragments thereof, peptidomimetics,that bind to the NC_(Ca-ATP) channel and either open the channel (e.g.,agonists) or block the channel (e.g., antagonists). For use in thetreatment of neural cell swelling or brain swelling, compounds thatblock the channel are preferred. Agonists that open or maintain thechannel in the open state include peptides, antibodies or fragmentsthereof, and other organic compounds that include the SUR1 subunit ofthe NC_(Ca-ATP) channel (or a portion thereof) and bind to and“neutralize” circulating ligand for SUR1.

With reference to screening of compounds that affect the NC_(Ca-ATP)channel, libraries of known compounds can be screened, including naturalproducts or synthetic chemicals, and biologically active materials,including proteins, for compounds which are inhibitors or activators.Preferably, such a compound is an NC_(Ca-ATP) antagonist, which includesan NC_(Ca-ATP) channel inhibitor, an NC_(Ca-ATP) channel blocker, a SUR1antagonist, SUR1 inhibitor, and/or a compound capable of reducing themagnitude of membrane current through the channel.

Compounds may include, but are not limited to, small organic orinorganic molecules, compounds available in compound libraries, peptidessuch as, for example, soluble peptides, including but not limited tomembers of random peptide libraries; (see, e.g., Lam, K. S. et al.,1991, Nature 354: 82-84; Houghten, R. et al., 1991, Nature 354: 84-86),and combinatorial chemistry-derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72: 767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope-bindingfragments thereof).

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able tocross the blood-brain barrier, gain entry into an appropriate neuralcell and affect the expression of the NC_(Ca-ATP) channel gene or someother gene involved in the NC_(Ca-ATP) channel activity (e.g., byinteracting with the regulatory region or transcription factors involvedin gene expression); or such compounds that affect the activity of theNC_(Ca-ATP) channel or the activity of some other intracellular factorinvolved in the NC_(Ca-ATP) channel activity.

To identify, make, generate, provide, manufacture or obtain modulator,one generally will determine the activity of the NC_(Ca-ATP) channel inthe presence, absence, or both of the candidate substance, wherein aninhibitor or antagonist is defined as any substance that down-regulates,reduces, inhibits, blocks or decreases the NC_(Ca-ATP) channelexpression or activity, and wherein an activator or agonist is definedas any substance that up-regulates, enhances, activates, increases oropens the NC_(Ca-ATP) channel. For example, a method may generallycomprise:

-   -   (a) providing a candidate substance suspected of activating or        inhibiting the NC_(Ca-ATP) channel expression or activity in        vitro or in vivo;    -   (b) assessing the ability of the candidate substance to activate        or inhibit the NC_(Ca-ATP) channel expression or activity in        vitro or in vivo;    -   (c) selecting an modulator; and    -   (d) manufacturing the modulator.

In certain embodiments, an alternative assessing step can be assessingthe ability of the candidate substance to bind specifically to theNC_(Ca-ATP) channel in vitro or in vivo;

In further embodiments, the NC_(Ca-ATP) channel may be provided in acell or a cell free system and the NC_(Ca-ATP) channel may be contactedwith the candidate substance. Next, the modulator is selected byassessing the effect of the candidate substance on the NC_(Ca-ATP)channel activity or expression. Upon identification of the modulator,the method may further provide manufacturing of the modulator.

V. METHODS OF CANCER TREATMENT

A. Treatment with an Agonist

In certain embodiments, the present invention is directed to a method oftreating a cancer patient in need of such treatment comprisingadministering an agonist of a NC_(Ca-ATP) channel of an neuronal cell ora neuroglia cell or a neural endothelial cell, wherein the agonistactivates a NC_(Ca-ATP) channel In specific embodiments, the agonisttargets a SUR1 of the NC_(Ca-ATP) channel. In certain embodiments, thecancer is located in the brain and, more specifically, comprises ametastatic tumor located in the brain.

Alternatively, the present invention is directed to a method ofdisrupting a tumor-brain barrier comprising administering an agonist ofa NC_(Ca-ATP) channel of an astrocyte, wherein said agonist activatessaid NC_(Ca-ATP) channel.

With the administration of an agonist of the NC_(Ca-ATP) channel, cellproliferation is abrogated, slowed, reduced or inhibited due to theopening of the NC_(Ca-ATP) channel. Such neuronal cells in which theagonist the NC_(Ca-ATP) channel may be administered may include any cellthat expresses SUR1.

An effective amount of an agonist or antagonist of NC_(Ca-ATP) channelthat may be administered to a cell includes a dose of about 0.0001 nM toabout 2000 μM. More specifically, doses of an agonist to be administeredare from about 0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM;about 0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5μM to about 2.0 μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about4.0 μM; about 4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about10 μM to about 50 μM; about 50 μM to about 100 μM; about 100 μM to about200 μM; about 200 μM to about 300 μM; about 300 μM to about 500 μM;about 500 μM to about 1000 μM; about 1000 μM to about 1500 μM and about1500 μM to about 2000 μM. Of course, all of these amounts are exemplary,and any amount in-between these points is also expected to be of use inthe invention.

It is envisioned that an agonist of NC_(Ca-ATP) channel orrelated-compound thereof will inhibit the proliferation of a cell orgrowth of a neoplasm, either directly or indirectly, by measurablyslowing, stopping, or reversing the growth rate of the cell or neoplasmor neoplastic cells in vitro or in vivo. Desirably, the growth rate isslowed by 20%, 30%, 50%, or 70% or more, as determined using a suitableassay for determination of cell growth rates.

Still further, the present invention provides methods for the treatmentof a cancer by administering an agonist of the NC_(Ca-ATP) channel. Theagonist or related-compound thereof can be administered parenterally oralimentary. Parenteral administrations include, but are not limited tointravenously, intradermally, intramuscularly, intraarterially,intrathecally, intraventricularly, intratumorally, subcutaneous, orintraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158;5,641,515; and 5,399,363 (each specifically incorporated herein byreference in its entirety). Alimentary administrations include, but arenot limited to orally, buccally, rectally, or sublingually.

The administration of the therapeutic compounds and/or the therapies ofthe present invention may include systemic, local and/or regional andmay oral, intravenous, and intramuscular. Alternatively, other routes ofadministration are also contemplated such as, for example, arterialperfusion, intracavitary, intraperitoneal, intrapleural,intraventricular, intratumoral, intraparenchyma and/or intrathecal. Ifdesired the therapeutic compound may be administered by the same routeas the chemotherapeutic agent, even if the therapeutic compound and thechemotherapeutic agent are not administered simultaneously. The skilledartisan is aware of determining the appropriate administration routeusing standard methods and procedures. In one example, where assessmentof a response to chemotherapy, both peripherally and centrally isdesired, the health care professional may use a systemic administration.

Treatment methods will involve treating an individual with an effectiveamount of a composition containing an agonist of NC_(Ca-ATP) channel orrelated-compound thereof. An effective amount is described, generally,as that amount sufficient to detectably and repeatedly to ameliorate,reduce, minimize or limit the extent of a disease or its symptoms. Morespecifically, it is envisioned that the treatment with the an agonist ofNC_(Ca-ATP) channel or related-compounds thereof will kill cells,inhibit cell growth, inhibit cell proliferation, inhibit metastasis,decrease tumor size and otherwise reverse or reduce the malignantphenotype of tumor cells, either directly or indirectly.

The effective amount of “therapeutically effective amounts” of the anagonist of NC_(Ca-ATP) channel or related-compounds thereof to be usedare those amounts effective to produce beneficial results, particularlywith respect to cancer treatment, in the recipient animal or patient.Such amounts may be initially determined by reviewing the publishedliterature, by conducting in vitro tests or by conducting metabolicstudies in healthy experimental animals. Before use in a clinicalsetting, it may be beneficial to conduct confirmatory studies in ananimal model, preferably a widely accepted animal model of theparticular disease to be treated. Preferred animal models for use incertain embodiments are rodent models, which are preferred because theyare economical to use and, particularly, because the results gained arewidely accepted as predictive of clinical value.

As is well known in the art, a specific dose level of active compoundssuch as an agonist of NC_(Ca-ATP) channel or related-compounds thereoffor any particular patient depends upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease undergoing therapy. The person responsible foradministration will determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

A therapeutically effective amount of an agonist of NC_(Ca-ATP) channelor related-compounds thereof as a treatment varies depending upon thehost treated and the particular mode of administration. In oneembodiment of the invention the dose range of the agonist of NC_(Ca-ATP)channel or related-compounds thereof will be about 0.0001 μg/kg bodyweight to about 500 mg/kg body weight. The term “ body weight ” isapplicable when an animal is being treated. When isolated cells arebeing treated, “ body weight ” as used herein should read to mean “totalcell body weight”. The term “total body weight” may be used to apply toboth isolated cell and animal treatment. All concentrations andtreatment levels are expressed as “body weight” or simply “kg” in thisapplication are also considered to cover the analogous “total cell bodyweight ” and “total body weight” concentrations. However, those of skillwill recognize the utility of a variety of dosage range, for example,0.0001 μg/kg body weight to 450 mg/kg body weight, 0.0002 μg/kg bodyweight to 400 mg/kg body weight, 0.0003 μg/kg body weight to 350 mg/kgbody weight, 0.0004 μg/kg body weight to 300 mg/kg body weight, 0.0005μg/kg body weight to 250 mg/kg body weight, 5.0 μg/kg body weight to 200mg/kg body weight, 10.0 μg/kg body weight to 150 mg/kg body weight,100.0 μg/kg body weight to 100 mg/kg body weight, or 1000 μg/kg bodyweight to 50 mg/kg body weight. Further, those of skill will recognizethat a variety of different dosage levels will be of use, for example,0.0001 μg/kg, 0.0002 μg/kg, 0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg,0.0007 μg/kg, 0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg,5.0 μg/kg, 10.0 μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80μg/kg, 90 μg/kg, 100 μg/kg, 120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg,180 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg,600 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 900 μg/kg, 1 mg/kg, 5 mg/kg,10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, and/or 30 mg/kg. Of course, allof these dosages are exemplary, and any dosage in-between these pointsis also expected to be of use in the invention. Any of the above dosageranges or dosage levels may be employed for an agonist of NC_(Ca-ATP)channel or related-compounds thereof.

Administration of the therapeutic agonist of NC_(Ca-ATP) channelcomposition of the present invention to a patient or subject will followgeneral protocols for the administration of chemotherapeutics, takinginto account the toxicity, if any, of the agonist of NC_(Ca-ATP)channel. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, aswell as surgical intervention, may be applied in combination with thedescribed therapy.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition (anagonist of NC_(Ca-ATP) channel or its related-compounds thereof)calculated to produce the desired responses in association with itsadministration, e.g., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime.

According to the present invention, one may treat the cancer by directlyinjection a tumor with an agonist of NC_(Ca-ATP) channel orrelated-compound composition. Alternatively, the tumor may be infused orperfused with the composition using any suitable delivery vehicle. Localor regional administration, with respect to the tumor, also iscontemplated. More preferably, systemic administration or oraladministration may be performed. Continuous administration also may beapplied where appropriate, for example, where a tumor is excised and thetumor bed is treated to eliminate residual, microscopic disease.Delivery via syringe or catheterization is preferred. Such continuousperfusion may take place for a period from about 1-2 hours, to about 2-6hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, toabout 1-2 wk or longer following the initiation of treatment. Generally,the dose of the therapeutic composition via continuous perfusion will beequivalent to that given by a single or multiple injections, adjustedover a period of time during which the perfusion occurs. For tumorsof >4 cm, the volume to be administered will be about 4-10 ml(preferably 10 ml), while for tumors of <4 cm, a volume of about 1-3 mlwill be used (preferably 3 ml). Multiple injections delivered as singledose comprise about 0.1 to about 1 ml volumes.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic agonist ofNC_(Ca-ATP) channel compositions may increase the resectability of thetumor due to shrinkage at the margins, either directly or indirectly, orby elimination of certain particularly invasive portions. Followingtreatments, resection may be possible. Additional treatments subsequentto resection will serve to eliminate microscopic residual disease at thetumor site.

B. Combined Cancer Therapy with an Agonist of NC_(Ca-ATP) Channel and/orOther Anticancer Agents

In the context of the present invention, it is contemplated that anagonist of NC_(Ca-ATP) channel or related-compounds thereof may be usedin combination with an additional therapeutic agent to more effectivelytreat cancer. Anticancer agents may include but are not limited to,radiotherapy, chemotherapy, gene therapy, hormonal therapy orimmunotherapy that targets cancer/tumor cells.

When an additional therapeutic agent is administered, as long as thedose of the additional therapeutic agent does not exceed previouslyquoted toxicity levels, the effective amounts of the additionaltherapeutic agent may simply be defined as that amount effective toinhibit and/or reduce the cancer growth when administered to an animalin combination with an agonist of NC_(Ca-ATP) channel orrelated-compounds thereof. This may be easily determined by monitoringthe animal or patient and measuring those physical and biochemicalparameters of health and disease that are indicative of the success of agiven treatment. Such methods are routine in animal testing and clinicalpractice.

To kill cells, induce cell-cycle arrest, inhibit cell growth, inhibitmetastasis, inhibit angiogenesis or otherwise reverse or reduce themalignant phenotype of cancer cells, either directly or indirectly,using the methods and compositions of the present invention, one wouldgenerally contact a cell with agonist of NC_(Ca-ATP) channel orrelated-compounds thereof in combination with an additional therapeuticagent. These compositions would be provided in a combined amounteffective to inhibit cell growth and/or induce apoptosis in the cell.This process may involve contacting the cells with agonist ofNC_(Ca-ATP) channel or related-compounds thereof in combination with anadditional therapeutic agent or factor(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes an agonist of NC_(Ca-ATP) channelor derivatives thereof and the other includes the additional agent.

Alternatively, treatment with an agonist of NC_(Ca-ATP) channel orrelated-compounds thereof may precede or follow the additional agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the additional agent is applied separately to the cell, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would contact the cell with bothmodalities within about 12-24 hr of each other and, more preferably,within about 6-12 hr of each other, with a delay time of only about 12hr being most preferred. In some situations, it may be desirable toextend the time period for treatment significantly, however, whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of either anagonist of NC_(Ca-ATP) channel or related-compounds thereof incombination with an additional therapeutic agent such as an anticanceragent will be desired. Various combinations may be employed, where anagonist of NC_(Ca-ATP) channel or related-compounds thereof is “A” andthe additional therapeutic agent is “B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

1. Chemotherapeutic Agents

In some embodiments of the present invention chemotherapy may beadministered, as is typical, in regular cycles. A cycle may involve onedose, after which several days or the weeks without treatment ensues fornormal tissues to recover from the drug's side effects. Doses may begiven several days in a row, or every other day for several days,followed by a period of rest. If more than one drug is used, thetreatment plan will specify how often and exactly when each drug shouldbe given. The number of cycles a person receives may be determinedbefore treatment starts (based on the type and stage of cancer) or maybe flexible, in order to take into account how quickly the tumor isshrinking. Certain serious side effects may also require doctors toadjust chemotherapy plans to allow the patient time to recover.

Chemotherapeutic agents that may be used in combination with agonists ofthe present invention or an related-compound thereof in the treatment ofcancer, include, but are not limited to cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil and methotrexate, or any related-compoundor derivative variant of the foregoing.

2. Radiotherapeutic Agents

Radiotherapeutic agents may also be use in combination with thecompounds of the present invention in treating a cancer. Such factorsthat cause DNA damage and have been used extensively include what arecommonly known as γ-rays, X-rays, and/or the directed delivery ofradioisotopes to tumor cells. Other forms of DNA damaging factors arealso contemplated such as microwaves and UV-irradiation. It is mostlikely that all of these factors effect a broad range of damage on DNA,on the precursors of DNA, on the replication and repair of DNA, and onthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

3. Immunotherapeutic Agents

Immunotherapeutics may also be employed in the present invention incombination with an agonist of NC_(Ca-ATP) channel or related-compoundsthereof in treating cancer Immunotherapeutics, generally, rely on theuse of immune effector cells and molecules to target and destroy cancercells. The immune effector may be, for example, an antibody specific forsome marker on the surface of a tumor cell. The antibody alone may serveas an effector of therapy or it may recruit other cells to actuallyeffect cell killing. The antibody also may be conjugated to a drug ortoxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin,pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells.

Generally, the tumor cell must bear some marker that is amenable totargeting, e.g., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

4. Inhibitors of Cellular Proliferation

The tumor suppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. The tumor suppressorsp53, p16 and C-CAM are described below.

High levels of mutant p53 have been found in many cells transformed bychemical carcinogenesis, ultraviolet radiation, and several viruses. Thep53 gene is a frequent target of mutational inactivation in a widevariety of human tumors and is already documented to be the mostfrequently mutated gene in common human cancers. It is mutated in over50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum ofother tumors.

The p53 gene encodes a 393-amino acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and areessential for the transforming ability of the oncogene. A single geneticchange prompted by point mutations can create carcinogenic p53. Unlikeother oncogenes, however, p53 point mutations are known to occur in atleast 30 distinct codons, often creating dominant alleles that produceshifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Winberg, 1991).

Another inhibitor of cellular proliferation is p16. The majortransitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G1. The activity of thisenzyme may be to phosphorylate Rb at late G1. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16INK4 has been biochemically characterized as a proteinthat specifically binds to and inhibits CDK4, and thus may regulate Rbphosphorylation (Serrano et al., 1993; Serrano et al., 1995). Since thep16INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of thisgene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16INK4 belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16B, p19, p21WAF1, and p27KIP1. The p16INK4 genemaps to 9p21, a chromosome region frequently deleted in many tumortypes. Homozygous deletions and mutations of the p16INK4 gene arefrequent in human tumor cell lines. This evidence suggests that thep16INK4 gene is a tumor suppressor gene. This interpretation has beenchallenged, however, by the observation that the frequency of thep16INK4 gene alterations is much lower in primary uncultured tumors thanin cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Okamoto et al., 1994; Arapet al., 1995). Restoration of wild-type p16INK4 function by transfectionwith a plasmid expression vector reduced colony formation by some humancancer cell lines (Okamoto et al., 1994; Arap et al., 1995).

Other genes that may be employed according to the present inventioninclude Rb, mda-7, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73,VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras,myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genesinvolved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF,or their receptors) and MCC.

5. Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process in cancertherapy (Kerr et al., 1972). The Bcl-2 family of proteins and ICE-likeproteases have been demonstrated to be important regulators andeffectors of apoptosis in other systems. The Bcl-2 protein, discoveredin association with follicular lymphoma, plays a prominent role incontrolling apoptosis and enhancing cell survival in response to diverseapoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Clearyet al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). Theevolutionarily conserved Bcl-2 protein now is recognized to be a memberof a family of related proteins, which can be categorized as deathagonists or death antagonists.

Members of the Bcl-2 that function to promote cell death such as Bax,Bak, Bik, Bim, Bid, Bad and Harakiri, are contemplated for use incombination with an agonist of NC_(Ca-ATP) channel or anrelated-compound thereof in treating cancer.

6. Surgery

It is further contemplated that a surgical procedure may be employed inthe present invention. Approximately 60% of persons with cancer willundergo surgery of some type, which includes preventative, diagnostic orstaging, curative and palliative surgery. Curative surgery includesresection in which all or part of cancerous tissue is physicallyremoved, excised, and/or destroyed. Tumor resection refers to physicalremoval of at least part of a tumor. In addition to tumor resection,treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and miscopically controlled surgery (Mohs' surgery). Itis further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

7. Other agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine related-compounds; or MIP-1, MIP-1beta, MCP-1,RANTES, and other chemokines. It is further contemplated that theupregulation of cell surface receptors or their ligands such as Fas/Fasligand, DR4 or DR5/TRAIL would potentiate the apoptotic inducingabilities of the present invention by establishment of an autocrine orparacrine effect on hyperproliferative cells. Increased intercellularsignaling by elevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyperproliferative efficacy of the treatments. Inhibitors ofcell adhesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

VI. METHODS OF CEREBRAL ISCHEMIA TREATMENT

A. Treatment with an Antagonist

In other embodiments, the therapeutic compound of the present inventioncomprises an antagonist of a NC_(Ca-ATP) channel of a neuronal cell, aneuroglial cell, a neural endothelial cell or a combination thereof.Antagonists are contemplated for use in treating adverse conditionsassociated with intracranial pressure and/or cytotoxic edema of thecentral nervous system. Such conditions include trauma (e.g., traumaticbrain injury (TBI)), ischemic brain injury, primary and secondaryneuronal injury, stroke, arteriovenous malformations (AVM),mass-occupying lesion (e.g., hematoma), and hemorrhagic infarction.Antagonists protect the cells expressing the NC_(Ca-ATP) channel, whichis desirable for clinical treatment in which ionic or cytotoxic edema isformed, in which capillary integrity is lost following ischemia, and inwhich gliotic capsule integrity is important and must be maintained toprevent the spread of infection, such as with a brain abscess. Those ofskill in the art realize that a brain abscess is a completely enclosedand results in cerebral swelling. The protection via inhibition of theNC_(Ca-ATP) channel is associated with a reduction in cerebral ionic andcytotoxic edema. Thus, the compound that inhibits the NC_(Ca-ATP)channel is neuroprotective.

In one aspect, the NC_(Ca-ATP) channel is blocked, inhibited, orotherwise is decreased in activity. In such examples, an antagonist ofthe NC_(Ca-ATP) channel is administered and/or applied. The antagonistmodulates the NC_(Ca-ATP) channel such that flux (ion and/or water)through the channel is reduced, ceased, decreased and/or stopped. Theantagonist may have a reversible or an irreversible activity withrespect to the activity of the NC_(Ca-ATP) channel of the neuronal cell,neuroglial cell, a neural endothelial cell or a combination thereof.Thus, inhibition of the NC_(Ca-ATP) channel can reduce cytotoxic edemaand death of endothelial cells which are associated with formation ofionic edema and with hemorrhagic conversion.

Accordingly, the present invention is useful in the treatment oralleviation of acute cerebral ischemia. According to a specificembodiment of the present invention the administration of effectiveamounts of the active compound can block the channel, which if remainedopen leads to neuronal cell swelling and cell death. A variety ofantagonists to SUR1 are suitable for blocking the channel. Examples ofsuitable SUR1 antagonists include, but are not limited to glibenclamide,tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole,LY397364, LY389382, glyclazide, glimepiride, estrogen, estrogenrelated-compounds and combinations thereof. In a preferred embodiment ofthe invention the SUR1 antagonists is selected from the group consistingof glibenclamide and tolbutamide. Another antagonist that can be used isMgADP. Still other therapeutic “strategies” for preventing neural cellswelling and cell death can be adopted including, but not limited tomethods that maintain the neural cell in a polarized state and methodsthat prevent strong depolarization.

In further embodiments, inhibitors or antagonist of the NC_(Ca-ATP)channel can be used to reduce or alleviate or abrogate hemorrhagicconversion. The pathological sequence that takes place in capillariesafter ischemia can be divided into 3 stages, based on the principalconstituents that move from the intravascular compartment into brainparenchyma (Ayata 2002; Betz, 1996; Betz 1989). The first stage ischaracterized by formation of “ionic” edema, during which the BBBremains intact, with movement of electrolytes (Na⁺, Cl⁻) plus water intobrain parenchyma. The second stage is characterized by formation of“vasogenic” edema, due to breakdown of the BBB, during whichmacromolecules plus water enter into brain parenchyma. The third stageis characterized by hemorrhagic conversion, due to catastrophic failureof capillaries, during which all constituents of blood extravasate intobrain parenchyma. In accordance with Starling's law, understanding thesephases requires that 2 things be identified: (i) the driving force that“pushes” things into parenchyma; and (ii) the permeability pore thatallows passage of these things into parenchyma.

Thus, the use of the antagonist or related-compounds thereof can reducethe mortality of a subject suffering from a stroke and/or rescue thepenumbra area or prevent damage in the penumbra area which comprisesareas of tissue that are at risk of becoming irreversibly damaged.

With the administration of an antagonist of the NC_(Ca-ATP) channel,endothelial cell depolarization is abrogated, slowed, reduced orinhibited due to the opening of the NC_(Ca-ATP) channel Thus, abrogationof cell depolarization results in abrogation or inhibition of Na influx,which prevents a change in osmotic gradient thereby preventing an influxof water into the endothelial cell and stopping cell swelling, blebbingand cytotoxic edema. Thus, preventing or inhibiting or attenuatingendothelial cell depolarization can prevent or reduce hemorrhagicconversion.

Neuronal cells in which the antagonist of the NC_(Ca-ATP) channel may beadministered may include any cell that expresses SUR1, for example anyneuronal cell, neuroglial cell or a neural endothelia cell.

Subjects that may be treated with the antagonist or related-compoundthereof include those that are suffering from or at risk of developingtrauma (e.g., traumatic brain injury (TBI)), ischemic brain injury,primary and secondary neuronal injury, stroke, arteriovenousmalformations (AVM), brain abscess, mass-occupying lesion, hemorrhagicinfarction, or any other condition associated with cerebral hypoxia orcerebral ischemia resulting in cerebral edema and/or increasedintracranial pressure, for example, but not limited to brain mass, brainedema, hematoma, end stage cerebral edema, encephalopathies, etc. Thus,the antagonist can be a therapeutic treatment in which the therapeutictreatment includes prophylaxis or a prophylactic treatment. Theantagonist or related-compounds thereof are neuroprotective.

Other subjects that may be treated with the antagonist of the presentinvention include those subjects that are at risk or predisposed todeveloping a stroke. Such subjects can include, but are not limited tosubjects that suffer from atrial fibrillations, clotting disorders,and/or risk of pulmonary emboli.

In certain embodiments, a subject at risk for developing a stroke mayinclude subjects undergoing treatments, for example, but not limited tocerebral/endovascular treatments, surgery (e.g., craniotomy, cranialsurgery, removal of brain tumors (e.g., hematoma), coronary arterybypass grafting (CABG), angiography, stent replacement, other vascularsurgeries, and/or other CNS or neurological surgeries), and treatment ofmyocardial infarction (MI) with thrombolytics. In such cases, thesubject may be treated with the antagonist or related-compound of thepresent invention prior to the actual treatment. Pretreatment caninclude administration of the antagonist and/or related-compound months(1, 2, 3, etc.), weeks (1, 2, 3, etc.), days (1, 2, 3, etc.), hours (1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), or minutes (15, 30, 60, 90, etc.)prior to the actual treatment or surgery. Treatment of the antagonistand/or related-compound can continue during the treatment and/or surgeryand after the treatment and/or surgery until the risk of developing astroke in the subject is decreased, lessened or alleviated.

In further embodiments, the antagonist of the present invention can begiven to a subject at risk of developing head/neck trauma, such as asubject involved in sports or other activities that have an increasedrisk of head/neck trauma.

An effective amount of an antagonist of the NC_(Ca-ATP) channel that maybe administered to a cell includes a dose of about 0.0001 nM to about2000 μM. More specifically, doses of an agonist to be administered arefrom about 0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM;about 0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5μM to about 2.0 μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about4.0 μM; about 4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about10 μM to about 50 μM; about 50 μM to about 100 μM; about 100 μM to about200 μM; about 200 μM to about 300 μM; about 300 μM to about 500 μM;about 500 μM to about 1000 μM; about 1000 μM to about 1500 μM and about1500 μM to about 2000 μM. Of course, all of these amounts are exemplary,and any amount in-between these points is also expected to be of use inthe invention.

The antagonist or related-compound thereof can be administeredparenterally or alimentary. Parenteral administrations include, but arenot limited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S.Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363(each specifically incorporated herein by reference in its entirety).Alimentary administrations include, but are not limited to orally,buccally, rectally, or sublingually.

The administration of the therapeutic compounds and/or the therapies ofthe present invention may include systemic, local and/or regionaladministrations, for example, topically (dermally, transdermally), viacatheters, implantable pumps, etc. Alternatively, other routes ofadministration are also contemplated such as, for example, arterialperfusion, intracavitary, intraperitoneal, intrapleural,intraventricular and/or intrathecal. The skilled artisan is aware ofdetermining the appropriate administration route using standard methodsand procedures. Other routes of administration are discussed elsewherein the specification and are incorporated herein by reference.

Treatment methods will involve treating an individual with an effectiveamount of a composition containing an antagonist of NC_(Ca-ATP) channelor related-compound thereof. An effective amount is described,generally, as that amount sufficient to detectably and repeatedly toameliorate, reduce, minimize or limit the extent of a disease or itssymptoms. More specifically, it is envisioned that the treatment withthe an antagonist of NC_(Ca-ATP) channel or related-compounds thereofwill inhibit cell depolarization, inhibit Na influx, inhibit an osmoticgradient change, inhibit water influx into the cell, inhibit cytotoxiccell edema, decrease stroke size, inhibit hemorrhagic conversion, anddecrease mortality of the subject.

The effective amount of an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof to be used are those amounts effective toproduce beneficial results, particularly with respect to stroketreatment, in the recipient animal or patient. Such amounts may beinitially determined by reviewing the published literature, byconducting in vitro tests or by conducting metabolic studies in healthyexperimental animals. Before use in a clinical setting, it may bebeneficial to conduct confirmatory studies in an animal model,preferably a widely accepted animal model of the particular disease tobe treated. Preferred animal models for use in certain embodiments arerodent models, which are preferred because they are economical to useand, particularly, because the results gained are widely accepted aspredictive of clinical value.

As is well known in the art, a specific dose level of active compoundssuch as an antagonist of the NC_(Ca-ATP) channel or related-compoundsthereof for any particular patient depends upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease undergoing therapy. The person responsible foradministration will determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

One of skill in the art realizes that the effective amount of theantagonist or related-compound thereof can be the amount that isrequired to achieve the desired result: reduction in the risk of stroke,reduction in intracranial pressure, reduction in cell death, reductionin stroke size, etc. This amount also is an amount that maintains areasonable level of blood glucose in the patient, for example, theamount of the antagonist maintains a blood glucose level of at least 60mmol/l, more preferably, the blood glucose level is maintain in therange of about 60 mmol/l to about 150 mmol/l. Thus, the amounts preventsthe subject from becoming hypoglycemic. If glucose levels are notnormal, then one of skill in the art would administer either insulin orglucose, depending upon if the patient is hypoglycemic or hyperglycemic.

An effective amount of an antagonist of the NC_(Ca-ATP) channel orrelated-compounds thereof as a treatment varies depending upon the hosttreated and the particular mode of administration. In one embodiment ofthe invention the dose range of the antagonist of the NC_(Ca-ATP)channel or related-compounds thereof will be about 0.01 μg/kg bodyweight to about 20,000 μg/kg body weight. The term “ body weight ” isapplicable when an animal is being treated. When isolated cells arebeing treated, “ body weight ” as used herein should read to mean “totalcell body weight”. The term “total body weight” may be used to apply toboth isolated cell and animal treatment. All concentrations andtreatment levels are expressed as “body weight” or simply “kg” in thisapplication are also considered to cover the analogous “total cell bodyweight” and “total body weight” concentrations. However, those of skillwill recognize the utility of a variety of dosage range, for example,0.01 μg/kg body weight to 20,000 μg/kg body weight, 0.02 μg/kg bodyweight to 15,000 μg/kg body weight, 0.03 μg/kg body weight to 10,000μg/kg body weight, 0.04 μg/kg body weight to 5,000 μg/kg body weight,0.05 μg/kg body weight to 2,500 μg/kg body weight, 0.06 μg/kg bodyweight to 1,000 μg/kg body weight, 0.07 μg/kg body weight to 500 μg/kgbody weight, 0.08 μg/kg body weight to 400 μg/kg body weight, 0.09 μg/kgbody weight to 200 μg/kg body weight or 0.1 μg/kg body weight to 100μg/kg body weight. Further, those of skill will recognize that a varietyof different dosage levels will be of use, for example, 0.0001 μg/kg,0.0002 μg/kg, 0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg, 0.0007 μg/kg,0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/kg,100 μg/kg, 120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg, 180 μg/kg, 200μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg,375 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 700μg/kg, 750 μg/kg, 800 μg/kg, 900 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 12mg/kg, 15 mg/kg, 20 mg/kg, and/or 30 mg/kg. Of course, all of thesedosages are exemplary, and any dosage in-between these points is alsoexpected to be of use in the invention. Any of the above dosage rangesor dosage levels may be employed for an antagonist of NC_(Ca-ATP)channel or related-compounds thereof.

Administration of the therapeutic antagonist of NC_(Ca-ATP) channelcomposition of the present invention to a patient or subject will followgeneral protocols for the administration of therapies used in stroketreatment, such as thrombolytics, taking into account the toxicity, ifany, of the antagonist of the NC_(Ca-ATP) channel. It is expected thatthe treatment cycles would be repeated as necessary. It also iscontemplated that various standard therapies, as well as surgicalintervention, may be applied in combination with the described therapy.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition (anantagonist of the NC_(Ca-ATP) channel or its related-compounds thereof)calculated to produce the desired responses in association with itsadministration, e.g., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime.

B. Combination Treatments

In the context of the present invention, it is contemplated that anantagonist of the NC_(Ca-ATP) channel or related-compounds thereof maybe used in combination with an additional therapeutic agent to moreeffectively treat a cerebral ischemic event, and/or decreaseintracranial pressure. In some embodiments, it is contemplated that aconventional therapy or agent, including but not limited to, apharmacological therapeutic agent may be combined with the antagonist orrelated-compound of the present invention.

Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an anticholesterol agent, anantiinflammatory agent, an antithrombotic/fibrinolytic agent,anticoagulant, antiplatelet, vasodilator, and/or diuretics.Thromoblytics that are used can include, but are not limited toprourokinase, streptokinase, and tissue plasminogen activator (tPA)Anticholesterol agents include but are not limited to HMG-CoA Reductaseinhibitors, cholesterol absorption inhibitors, bile acid sequestrants,nicotinic acid and derivatives thereof, fibric acid and derivativesthereof. HMG-CoA Reductase inhibitors include statins, for example, butnot limited to atorvastatin calcium (Lipitor®), cerivastatin sodium(Baycol®), fluvastatin sodium (Lescol®), lovastatin (Advicor®),pravastatin sodium (Pravachol®), and simvastatin (Zocor®). Agents knownto reduce the absorption of ingested cholesterol include, for example,Zetia®. Bile acid sequestrants include, but are not limited tocholestryramine, cholestipol and colesevalam. Other anticholesterolagents include fibric acids and derivatives thereof (e.g., gemfibrozil,fenofibrate and clofibrate); nicotinic acids and derivatives thereof(e.g., nician, lovastatin) and agents that extend the release ofnicotinic acid, for example niaspan. Antiinflammatory agents include,but are not limited to non-sterodial anti-inflammatory agents (e.g.,naproxen, ibuprofen, celeoxib) and sterodial anti-inflammatory agents(e.g., glucocorticoids). Anticoagulants include, but are not limited toheparin, warfarin, and coumadin. Antiplatelets include, but are notlimited to aspirin, and aspirin related-compounds, for exampleacetaminophen. Diuretics include, but are not limited to such asfurosemide (Lasix®), bumetanide (Bumex®), torsemide (Demadex®), thiazide& thiazide-like diuretics (e.g., chlorothiazide (Diuril®) andhydrochlorothiazide (Esidrix®), benzthiazide, cyclothiazide, indapamide,chlorthalidone, bendroflumethizide, metolazone), amiloride, triamterene,and spironolacton. Vasodilators include, but are not limited tonitroglycerin,

Thus, in certain embodiments, the present invention comprisesco-administration of an antagonist of the NC_(Ca-ATP) channel with athrombolytic agent. Co-administration of these two compounds willincrease the therapeutic window of the thrombolytic agent. Examples ofsuitable thrombolytic agents that can be employed in the methods andpharmaceutical compositions of this invention are prourokinase,streptokinase, and tissue plasminogen activator (tPA).

In certain embodiments, the present invention comprisesco-administration of an antagonist of the NC_(Ca-ATP) channel withglucose or related carbohydrate to maintain appropriate levels of serumglucose. Appropriate levels of blood glucose are within the range ofabout 60 mmol/l to about 150 mmol/liter. Thus, glucose or a relatedcarbohydrate is administered in combination to maintain the serumglucose within this range.

When an additional therapeutic agent, as long as the dose of theadditional therapeutic agent does not exceed previously quoted toxicitylevels, the effective amounts of the additional therapeutic agent maysimply be defined as that amount effective to reduce cerebral edema whenadministered to an animal in combination with an agonist of NC_(Ca-ATP)channel or related-compounds thereof. This may be easily determined bymonitoring the animal or patient and measuring those physical andbiochemical parameters of health and disease that are indicative of thesuccess of a given treatment. Such methods are routine in animal testingand clinical practice.

To inhibit hemorrhagic conversion, reduce cell swelling, etc., using themethods and compositions of the present invention, one would generallycontact a cell with antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof in combination with an additional therapeuticagent, such as tPA, aspirin, statins, diuretics, warfarin, coumadin,mannitol, etc. These compositions would be provided in a combined amounteffective to inhibit hemorrhagic conversion, cell swelling and edema.This process may involve contacting the cells with agonist ofNC_(Ca-ATP) channel or related-compounds thereof in combination with anadditional therapeutic agent or factor(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes an antagonist of the NC_(Ca-ATP)channel or derivatives thereof and the other includes the additionalagent.

Alternatively, treatment with an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof may precede or follow the additional agenttreatment by intervals ranging from minutes to hours to weeks to months.In embodiments where the additional agent is applied separately to thecell, one would generally ensure that a significant period of time didnot expire between the time of each delivery, such that the agent wouldstill be able to exert an advantageously combined effect on the cell. Insuch instances, it is contemplated that one would contact the cell withboth modalities within about 1-24 hr of each other and, more preferably,within about 6-12 hr of each other.

Typically, for maximum benefit of the thrombolytic agent, or therapymust be started within three hours of the onset of stroke symptoms,making rapid diagnosis and differentiation of stroke and stroke typecritical. However, in the present invention, administration of theNC_(Ca-ATP) channel with a thrombolytic agent increases this therapeuticwindow. The therapeutic window for thrombolytic agents may be increasedby several (4-8) hours by co-administering antagonist of the NC_(Ca-ATP)channel.

Yet further, the combination of the antagonist and tPA results in adecrease or prevention of hemorrhagic conversion following reperfusion.Hemorrhagic conversion is the transformation of a bland infarct into ahemorrhagic infarct after restoration of circulation. It is generallyaccepted that these complications of stroke and of reperfusion areattributable to capillary endothelial cell dysfunction that worsens asischemia progresses. Thus, the present invention is protective of theendothelial cell dysfunction that occurs as a result of an ischemicevent.

Endothelial cell dysfunction comprises three phases. Phase one ischaracterized by formation of ionic edema with the blood brain barrierstill intact. The second phase is characterized by formation ofvasogenic edema in which the blood brain barrier is no longer intact.Phase three is characterized by hemorrhagic conversion due to failure ofcapillary integrity during which all constituents of blood, includingerythrocytes, extravasate into brain parenchyma. Disruption of BBBinvolves ischemia-induced activation of endothelial cells that resultsin expression and release of MMPs, specifically, MMP-2 (gelatinase A)and MMP-9 (gelatinase B).

Since hemorrhagic conversion increases mortality of the patient, it isessential that these patients receive treatment in an urgent manner Forexample, it is known that hemorrhagic conversion typically results inpatients if reperfusion and tPA treatment is delayed beyond 3 hr or moreafter thrombotic stroke. Thus, the administration of the antagonist ofthe present invention will reduce necrotic death of ischemic endothelialcells, and will thereby prolong the therapeutic window for tPA, therebydecreasing mortality of the patient.

VII. DIAGNOSTICS

The antagonist or related-compound can be used for diagnosing,monitoring, or prognosis of ischemia or damage to neurons, glial cellsor in monitoring neuronal cells in zones of cerebral edema, metastatictumors, etc.

A. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingexpression of any portion of a NC_(Ca-ATP) channel, for example,expression of the regulatory unit, SUR1, and/or expression of thepore-forming subunit. This may comprise determining the level of SUR1expressed and/or the level of the pore-forming subunit expressed. It isunderstood by the present invention that the up-regulation or increasedexpression of the NC_(Ca-ATP) channel relates to increased levels ofSUR1, which correlates to increased neuronal damage, such as cerebraledema.

Firstly, a biological sample is obtained from a subject. The biologicalsample may be tissue or fluid. In certain embodiments, the biologicalsample includes cells from the brain and/or cerebral endothelial cellsor microvessels and/or gliotic capsule. For example, in metastatictumors, glial cells are activated and form a capsule around the tumor.

Nucleic acids used are isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementary DNA(cDNA). In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given subjectwith a statistically significant reference group of normal subjects andsubjects that have been diagnosed with a stroke, cancer, cerebral edema,etc.

Yet further, it is contemplated that chip-based DNA technologies such asthose described by Hacia et al., (1996) and Shoemaker et al., (1996) canbe used for diagnosis. Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al., (1994); Fodor et al., (1991).

B. Other types of diagnosis

In order to increase the efficacy of molecules, for example, compoundsand/or proteins and/or antibodies, as diagnostic agents, it isconventional to link or covalently bind or complex at least one desiredmolecule or moiety.

Certain examples of conjugates are those conjugates in which themolecule (for example, protein, antibody, and/or compound) is linked toa detectable label. “Detectable labels” are compounds and/or elementsthat can be detected due to their specific functional properties, and/orchemical characteristics, the use of which allows the antibody to whichthey are attached to be detected, and/or further quantified if desired.

Conjugates are generally preferred for use as diagnostic agents.Diagnostics generally fall within two classes, those for use in in vitrodiagnostics, such as in a variety of immunoassays, and/or those for usein vivo diagnostic protocols, generally known as “molecule-directedimaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to molecules, for example, antibodies (see, for e.g.,U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporatedherein by reference). The imaging moieties used can be paramagneticions; radioactive isotopes; fluorochromes; NMR-detectable substances;X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹¹carbon, ¹⁴carbon,⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷,³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron,³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur,technicium^(99m) and/or yttrium⁹⁰. ¹²⁵I is often being preferred for usein certain embodiments, and technicium^(99m) and/or indium¹¹¹ are alsooften preferred due to their low energy and suitability for long rangedetection.

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of conjugates contemplated in the present invention arethose intended primarily for use in vitro, where the molecule is linkedto a secondary binding ligand and/or to an enzyme (an enzyme tag) thatwill generate a colored product upon contact with a chromogenicsubstrate. Examples of suitable enzymes include urease, alkalinephosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.Preferred secondary binding ligands are biotin and/or avidin andstreptavidin compounds. The use of such labels is well known to those ofskill in the art and are described, for example, in U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241; each incorporated herein by reference.

The steps of various other useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Nakamura et al.,(1987) Immunoassays, in their most simple and direct sense, are bindingassays. Certain preferred immunoassays are the various types ofradioimmunoassays (RIA) and immunobead capture assay.Immunohistochemical detection using tissue sections also is particularlyuseful. However, it will be readily appreciated that detection is notlimited to such techniques, and Western blotting, dot blotting, FACSanalyses, and the like also may be used in connection with the presentinvention.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary molecules/antibodies against the SUR1 orregulatory subunit of the Na_(CaATP) channel are considered to be ofparticular use in this regard. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody or abiotin/avidin ligand binding arrangement, as is known in the art.

In addition to the above imaging techniques, one of skill in the art isalso aware that positron emission tomography, PET imaging or a PET scan,can also be used as a diagnostic examination. PET scans involves theacquisition of physiologic images based on the detection of radiationfrom the emission of positrons. Positrons are tiny particles emittedfrom a radioactive substance administered to the subject.

Thus, in certain embodiments of the present invention, the antagonist orrelated-compound thereof is enzymatically-, radiolabel-, orfluorescently-tagged, as described above and used to diagnosis, monitor,and/or stage neuronal damage by cerebral edema. For example, theenzymatically-, radiolabel-, or fluorescently-tagged antagonist orrelated-compound thereof can be used to determine the size, limitsand/or boundaries of tumors. It is difficult to determine the boundariesof certain tumors, for example, metastatic tumors. In metastatic tumors,glial cells are activated and form a capsule or gliotic capsule aroundthe tumor. Thus, the labeled antagonist or related-compound thereof canbe used to determine the border of tumor, which can enhance theefficiency of its removal by the surgeon. Still further, the labeledantagonist or related-compound thereof may be used to determine ordefine the penumbra or the areas at risk for later infarction or damageafter a stroke.

VIII. FORMULATIONS AND ROUTES FOR ADMINISTRATION OF COMPOUNDS

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more modulators of NC_(Ca-ATP) channel(antagonist and/or agonist) or related-compounds or additional agentdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of anpharmaceutical composition that contains at least one modulators ofNC_(Ca-ATP) channel (antagonist and/or agonist) or related-compounds oradditional active ingredient will be known to those of skill in the artin light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The modulators of NC_(CaATP) channel (antagonist and/or agonist) orrelated-compounds may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraventricularly,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The modulators of NC_(Ca-ATP) channel (antagonist and/or agonist) orrelated-compounds may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include modulators ofNC_(CaATP) channel (antagonist and/or agonist) or related-compounds, oneor more lipids, and an aqueous solvent. As used herein, the term “lipid”will be defined to include any of a broad range of substances that ischaracteristically insoluble in water and extractable with an organicsolvent. This broad class of compounds are well known to those of skillin the art, and as the term “lipid” is used herein, it is not limited toany particular structure. Examples include compounds which containlong-chain aliphatic hydrocarbons and their derivatives. A lipid may benaturally occurring or synthetic (i.e., designed or produced by man)However, a lipid is usually a biological substance. Biological lipidsare well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the modulators of NC_(CaATP) channel (antagonistand/or agonist) or related-compounds may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeutic and/orprophylatic interventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according tot he response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

A. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the modulators ofNC_(CaATP) channel (antagonist and/or agonist) or related-compounds areformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, modulators of NC_(CaATP) channel (antagonistand/or agonist) or related-compounds may be administered via aparenteral route. As used herein, the term “parenteral” includes routesthat bypass the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered for example, but notlimited to intravenously, intradermally, intramuscularly,intraarterially, intraventricularly, intrathecally, subcutaneous, orintraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468,5,543,158; 5,641,515; and 5,399,363 (each specifically incorporatedherein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, DMSO, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compoundmodulators of NC_(CaATP) channel (antagonist and/or agonist) orrelated-compounds may be formulated for administration via variousmiscellaneous routes, for example, topical (i.e., transdermal)administration, mucosal administration (intranasal, vaginal, etc.)and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

IX. DIAGNOSTIC OR THERAPEUTIC KITS

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, it is envisioned that a compound that selectivelybinds to or identifies SUR1 may be comprised in a diagnositc kit. Suchcompounds can be referred to as an “SUR1 marker”, which may include, butare not limited to antibodies (monoclonal or polyclonal), SUR1oligonucleotides, SUR1 polypeptides, small molecule or combinationsthereof, antagonist, agonist, etc. It is envisioned that any of theseSUR1 markers may be linked to a radioactive substance and/or afluorescent marker and/or a enzymatic tag for quick determination. Thekits may also comprise, in suitable container means a lipid, and/or anadditional agent, for example a radioactive or enzymatic or florescentmarker.

The kits may comprise a suitably aliquoted SUR1 marker, lipid and/oradditional agent compositions of the present invention, whether labeledor unlabeled, as may be used to prepare a standard curve for a detectionassay. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the SUR1 marker, lipid,additional agent, and any other reagent containers in close confinementfor commercial sale. Such containers may include injection or blowmolded plastic containers into which the desired vials are retained.

Therapeutic kits of the present invention are kits comprising anantagonist, agonist or an related-compound thereof. Depending upon thecondition and/or disease that is being treated, the kit may comprise anSUR1 antagonist or related-compound thereof to block and/or inhibit theNC_(CaATP) channel or the kit may comprise an SUR1 agonist orrelated-compound thereof to open the NC_(CaATP) channel Such kits willgenerally contain, in suitable container means, a pharmaceuticallyacceptable formulation of SUR1 antagonist, agonist or related-compoundthereof. The kit may have a single container means, and/or it may havedistinct container means for each compound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The SUR1 antagonist,agonist or related-compounds thereof may also be formulated into asyringeable composition. In which case, the container means may itselfbe a syringe, pipette, and/or other such like apparatus, from which theformulation may be applied to an infected area of the body, injectedinto an animal, and/or even applied to and/or mixed with the othercomponents of the kit.

Examples of aqueous solutions include, but are not limited to ethanol,DMSO and/or Ringer's solution. In certain embodiments, the concentrationof DMSO or ethanol that is used is no greater than 0.1% or (1 ml/1000L).

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which the SUR1antagonist, agonist or related-compounds thereof is suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of the SUR1antagonist, agonist or related-compounds thereof within the body of ananimal. Such an instrument may be a syringe, pipette, forceps, and/orany such medically approved delivery vehicle.

In addition to the SUR1 antagonist, agonist or related-compoundsthereof, the kits may also include a second active ingredient. Examplesof the second active ingredient include substances to preventhypoglycemia (e.g., glucose, D5W, glucagon, etc.), thrombolytic agents,anticoagulants, antiplatelets, statins, diuretics, vasodilators, etc.These second active ingredients may be combined in the same vial as theSUR1 antagonist, agonist or related-compounds thereof or they may becontained in a separate vial.

Still further, the kits of the present invention can also includeglucose testing kits. Thus, the blood glucose of the patient is measuredusing the glucose testing kit, then the SUR1 antagonist, agonist orrelated-compounds thereof can be administered to the subject followed bymeasuring the blood glucose of the patient.

In addition to the above kits, the therapeutic kits of the presentinvention can be assembled such that an IV bag comprises a septum orchamber which can be opened or broken to release the compound into theIV bag. Another type of kit may include a bolus kit in which the boluskit comprises a pre-loaded syringe or similar easy to use, rapidlyadministrable device. An infusion kit may comprise the vials or ampoulesand an IV solution (e.g., Ringer's solution) for the vials or ampoulesto be added prior to infusion. The infusion kit may also comprise abolus kit for a bolus/loading dose to be administered to the subjectprior, during or after the infusion.

X. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Modulation by Estrogen

A characteristic feature of K_(ATP) channels (Kir6.1, Kir6.2) is thatchannel affinity for ATP is modulated by the presence of the membranelipid, PIP₂. The open-state stability of K_(ATP) channels is increasedby application of PIP₂ to the cytoplasmic side of the membrane(Ashcroft, 1998; Baukrowitz et al., 1998; Rohacs et al., 1999). Anincrease in the open-state stability is manifested as an increase in thechannel open probability in the absence of ATP, and in a correspondingdecrease in sensitivity to inhibition by ATP (Enkvetchakul et al., 2000;Haruna et al., 2000; Koster et al., 1999; and Larsson et al., 2000).

Given the numerous similarities between the K_(ATP) channel and theNC_(Ca-ATP) channel, the inventors postulated that ATP-sensitivity ofthe NC_(Ca-ATP) channel would respond to PIP₂ in the same way. This wastested by studying NC_(Ca-ATP) channels in inside out patches with Cs⁺as the charge carrier, and with 1 μM Ca²⁺ and 10 μM ATP in the bath,with the latter expected to fully block the channel Under theseconditions, only the NC_(Ca-ATP) channel was recorded in R1 astrocytes.When PIP₂ (50 μM) was added to the bath, channel activity becameprominent (FIG. 1), as predicted by analogy to the effect of PIP₂ onK_(ATP) channels. This channel activity was blocked by glibenclamide,confirming identity of the channel.

To determine if a receptor-mediated mechanism was involved in themodulation of NC_(Ca-ATP) channel activity, a well known phospholipase C(PLC) was used to study if PLC activation would cause degradation andconsumption of PIP₂ and thereby increase affinity for ATP, e.g., reducechannel opening. Estrogen is a well known PLC activator in brain as wellas elsewhere (Beyer et al., 2002; Le Mellay et al., 1999; Qui et al.,2003). For this experiment, cell attached patches were studied toprevent alteration of intracellular signaling machinery. NC_(Ca-ATP)channel activity was produced by exposure to Na azide to cause depletionof cellular ATP (FIG. 2, initial part of the record).

When estrogen (E2; 10 nM) was applied to the bath, activity due to theNC_(Ca-ATP) channel was soon terminated (FIG. 2). This suggested thatestrogen exerted regulatory control over the NC_(Ca-ATP) channel, andsuggested that an estrogen receptor capable of rapid (non-genomic)activation of signaling cascades was present on these cells.

Next, to determine whether estrogen receptors could be detected in R1astrocytes from males and females. Gelatin sponge implants wereharvested 7 days after implantation in a group of 3 female rats (F) andanother group of 3 male rats (M). Pooled protein from each group wasanalyzed at 2 dilutions (4×=50 μg total protein; 1×=12.5 μg totalprotein) by Western blotting, with protein from uterus being used as acontrol (FIG. 3A). Membranes were blotted with an antibody thatrecognized both α and β estrogen receptors. Both males and femalesshowed prominent bands at the appropriate molecular weights for the α(66 kDa) and β (55 kDa) receptors (FIG. 3) (Hiroi et al., 1999). Thesame samples of protein from males and females were also used to confirmpresence of SUR1, with protein from pancreas used as a positive control(FIG. 3B). Notably, estrogen receptors have previously been reported inastrocytes from males and females (Choi et al., 2001). In cerebralcortex, the β isoform is reportedly more abundant (Guo et al., 2001) assuggested by the Western blot.

Next, the electrophysiological experiment of FIG. 2 was repeated usingR1 astrocytes harvested from male rats. As above, cell attached patcheswere studied in which NC_(Ca-ATP) channel activity was activated bydepletion of intracellular ATP following exposure to Na azide (FIG. 4A).Examination of the record at higher temporal resolution confirmedactivity of a well defined channel of the appropriate conductance forthe NC_(Ca-ATP) channel (FIG. 4B). When estrogen was applied to the bath(FIG. 4, E2, 10 nM, arrow), activity due to the NC_(Ca-ATP) channel wasquickly terminated (FIG. 4). These data provided further evidence thatestrogen exerted regulatory control over the NC_(Ca-ATP) channel, andsuggested, in addition, that this response was equally robust in R1astrocytes from males and females.

By analogy to the effects of estrogen, other mechanisms that depletePIP₂, including other receptor-mediated mechanism as well as more directactivators of PLC such as G-proteins etc., would be expected to have asimilar inhibitory effect on activity of the NC_(Ca-ATP) channel andthereby exert a protective effect.

Example 2 The Gliotic Capsule

The standard model involved placing a stab injury into the parietal lobeof an anesthetized rat and implanting a sterile foreign body (gelatinsponge; Gelfoam®) into the stab wound. Variants of the standard modelincluded impregnating the sponge with a substance (e.g.,lipopolysaccharide, LPS) or infusing a substance continuously in vivousing an osmotic mini-pump with the delivery catheter placed directlyinto the sponge. The injury procedure was well tolerated by the animals,with virtually no morbidity or mortality and minimal pain. After anappropriate time in vivo, the whole brain was harvested for histologicalor immunohistochemical study of tissue sections. Alternatively, if thesponge itself was gently removed from the brain, the inner zone of thegliotic capsule adheres to the sponge and was excised along with it.Thus, the sponge was assayed for protein (e.g., Western) or mRNA(RT-PCR), or it was enzymatically dissociated to yield constituent cellsfor electrophysiological or other single-cell measurements.

The gliotic capsule was well developed 7-10 days after injury. Thegliotic capsule was visualized in coronal sections by perfusing theanimal with Evans Blue prior to perfusion-fixation of the brain (FIG.5A). A region of edema (dark) was seen to outline the avascular glioticcapsule (light) that surrounded the gelatin sponge (dark)Immunohistochemical examination with anti-GFAP antibodies showed thatthe brain parenchyma in the vicinity of the sponge harbors manyGFAP-positive reactive astrocytes (FIG. 5B; arrow showed where thegelatin sponge was). At higher power, these intraparenchymalGFAP-positive cells were shown to be large and to bear many prominentcell processes (FIG. 5C, arrow). Examining the gelatin sponge itselfshowed GFAP-positive reactive astrocytes that migrated into theinterstices of the sponge (FIG. 5D, arrow).

Example 3 Isolation of Cells from the Gliotic Capsule

Phase contrast microscopy of cells freshly isolated by papain digestionof the inner zone of the gliotic capsule and gelatin sponge revealedthree types of cells. Most of the cells (>90%) were large, round, haveno cell processes and were phase-bright (FIG. 6A). A number of cells(3-5%) were small, round, have no cell processes and were phase-dark(FIG. 6B). Occasionally, a cell was found that was intermediate in size,was phase-bright and had multiple processes that were more than one celldiameter in length (Chen et al., 2003). Immunofluorescence study showedthat all of these cells were strongly positive for typical astrocytemarkers, including GFAP (FIG. 6C,D) and vimentin (FIG. 6E,F). Microgliawere not prominent in the inner zone of the gliotic capsule itself, asindicated by sparse labeling for OX-42. Cells of the inner zone of thegliotic capsule were negative for the 02A progenitor marker, A2B5, andthe fibroblast marker, prolyl 4-hydroxylase (Dalton et al., 2003).

As with freshly isolated cells, three morphologically distinct types ofcells were observed in primary culture. Most cells (>90%) were largepolygonal cells (FIG. 6Gb), a few (3-5%) were small bipolar cells (FIG.6Ga), and only occasionally were process-bearing stellate-shaped cellsobserved (Perillan et al., 2000). All of these cells were stronglylabeled with anti-GFAP antibodies (FIG. 6H). Experiments in which cellsobtained by enzymatic digestion were followed individually in primaryculture showed that the large phase-bright cells develop into largepolygonal cells (FIG. 6Gb), and the small phase-dark cells developedinto small bipolar cells (FIG. 6Ga) (Dalton et al., 2003).

The three morphologically distinguishable types of GFAP-positiveastrocytes from the inner zone of the gliotic capsule exhibited verydifferent macroscopic whole cell electrophysiological profiles:

(i) Electrophysiological studies on stellate astrocytes showed that theyexpressed Kir2.3 and Kir4.1 inward rectifier channels, andimmunolabeling experiments suggested that they also expressed K_(ATP)channels comprised of SUR1 and Kir6.1 subunits (Chen et al., 2003;Perillan et al., 2000);

(ii) Electrophysiological studies on R2 astrocytes showed that theyexpressed a novel Ca²⁺-activated Cl-channel that was sensitive to thepolypeptide toxin from the scorpion, Leiurus quinquestriatus (Dalton etal., 2003). Only the R2 astrocyte expressed this channel.

(iii) Electrophysiological studies on R1 astrocytes showed that theyexpress Kir2.3 inward rectifier channels that are regulated by TGFβ1 viaPKCδ (Perillan et al., 2002; Perillan et al., 2000). When freshlyisolated but not after culturing, R1 astrocytes also expressd a novelSUR1-regulated NC_(Ca-ATP) channel (Chen et al., 2003; Chen et al.,2001).

Example 4 Expression of SUR1

Glibenclamide binds to sulfonylurea receptors, SUR1 and SUR2, withhigher affinity for SUR1. Immunofluorescence studies were performedusing anti-SURx antibodies. The inner zone of the gliotic capsuleimmediately outside of the gelatin sponge (gf in FIG. 7) was stronglylabeled with anti-SUR1 antibody (FIG. 8A) but not with anti-SUR2antibody (FIG. 7B). Although individual cells were not discerned at lowmagnification, higher magnification showed that SUR1 label was uniformlydistributed in individual cells after isolation (FIG. 7C).

Evidence for transcription of SUR1, but not SUR2 was also found inRT-PCR experiments run on mRNA from gelatin sponges isolated 7 daysafter implantation. The signal observed in astrocytes (FIG. 7D, lane 3)was present at the appropriate position on the gel, similar to that fromcontrol insulinoma RIN-m5f cells (FIG. 7D, lane 2). By contrast, mRNAfor SUR2 is not transcribed in reactive astrocytes (FIG. 7D, lane 5)although it is in cardiomyocytes used as control (FIG. 7D, lane 4).

Example 5 Characterization of the Inner Zone of the Gliotic Capsule

To examine whether or not all GFAP-positive reactive astrocytes in thegliotic capsule are SUR1 positive, brains from rats that had beenimplanted 1 week earlier with a gelatin sponge, then perfusion-fixed andequilibrated in 40% sucrose in PBS ×2 days were studied. Cryostatsections were double labeled with anti-GFAP and anti-SUR1 antibodies andstudied with immunofluorescence. For this and other immunolabelingexperiments, standard control protocol included use of the appropriateimmunogenic peptide when available or omission of primary antibody.

Five animals were sectioned and imaged with low power images. The imagesinvariably showed that the depth (thickness) of the GFAP response fromthe edge of the gelatin sponge was several-fold greater than the depthof the SUR1 response. Measurements of the depth of the GFAP responseyielded values of about 400-500 μm (FIG. 8A; in FIGS. 8A-8I, thelocation of the gelatin sponge implant was always to the left; bar inFIG. 8F equals 100 μm). By contrast, the prominent portion of the SUR1response extended for a depth of only 25-50 μm (FIG. 8D). Outside of theSUR1-positive zone was a wide region of GFAP-positive reactiveastrocytes that were mostly SUR1 negative. The SUR1 response was alwayslocated precisely at the interface with the foreign body, in theinnermost zone of the gliotic capsule. Cells that were SUR1 positivewere always GFAP positive. It was evident from this experiment thatcells clinging to the gelatin sponge and that were harvested with itwere likeliest to express SUR1. Also, it was clear that R1 astrocytes inthis innermost region comprised a unique subpopulation of reactiveastrocytes. From this observation emerged the concept of the “innerzone” of the gliotic capsule as being a unique entity, distinct from theremainder of the gliotic capsule.

Example 6 Other Characteristics of the Inner Zone of the Gliotic Capsule

Other studies were performed to further evaluate the inner zone of thegliotic capsule. In previous experiments, it was found that primaryculture of R1 astrocytes under normoxic culture conditions resulted inloss of the SUR1-regulated NC_(Ca-ATP) channel after 3 days, whereascultured under hypoxic conditions resulted in continued expression ofthe channel (Chen et al., 2003). Thus, it was determined that expressionof the channel required hypoxic conditions, and thus the inner zone ofthe gliotic capsule where SUR1 expressing R1 astrocytes were found mightalso be hypoxic. To evaluate this, the histochemical marker,pimonidazole, was used which at pO₂<10 mm Hg, forms irreversiblecovalent adducts with cellular proteins that can be detectedimmunohistochemically (Arteel et al, 1998; Hale et al., 2002; Kennedy etal., 1997).

Briefly, rats were prepared with a stab injury and implantation of agelatin sponge. Rats were allowed to survive 1 week. Pimonidazole wasadministered prior to death, and cryosections were processed forimmunofluorescence study using the appropriate antibody to detectpimonidazole adducts. Cryosections were double labeled for GFAP. Thisexperiment confirmed the presence of hypoxic conditions restricted tothe SUR1-positive inner zone of the gliotic capsule, with the mostprominent pimonidazole labeling extending only 20-50 μm deep (FIG. 8B;GFAP not shown but the depth of the GFAP response resembled that in FIG.8A). High resolution imaging showed that pimonidazole labeling (FIG. 8G,upper right) was present in large GFAP-positive astrocytes (FIG. 8G,lower left).

It was reasoned that hypoxia of the inner zone might lead toup-regulation/activation of the hypoxia-responsive transcription factor,HIF-1. To examine this, immunolabeling was performed of sections withanti-HIF-1α antibodies with co-labeling for GFAP. This experimentconfirmed that HIF-1α labeling was mostly restricted to theSUR1-positive inner zone of the gliotic capsule, with labeling extendingonly 20-50 μm deep (FIG. 8C; GFAP not shown but the depth of the GFAPresponse resembled that in FIG. 8A). High resolution imaging showed thatHIF-1α labeling (FIG. 8H, upper right) was present in largeGFAP-positive astrocytes (FIG. 8H, lower left).

Expression of tight junction proteins was also examined. Two tightjunction proteins, ZO-1 and occludin-5, were studied, labeling alternatecryosections with antibodies directed against these proteins. Sectionswere double labeled for GFAP. Again, only the innermost layer 20-50 μmdeep was labeled for either ZO-1 or occludin-5 (FIG. 8E and 8F; GFAP notshown but the depth of the GFAP response resembled that in FIG. 8A).High resolution imaging showed that occludin-5 labeling (FIG. 8I, upperright) was present in large GFAP-positive astrocytes (FIG. 8I, lowerleft).

Thus, the inner zone of the gliotic capsule, with its R1 astrocytes thatexpress SUR1-regulated NC_(Ca-ATP) channels and tight junction proteins,may be acting as an important barrier between the foreign body and thebrain, e.g., a foreign body-brain barrier (FbBB). If true, one wouldexpect that breaching the barrier might significantly affect the overallresponse to injury.

Example 7 Manipulation of the Inner Zone

Rats were prepared with a stab injury and implantation of a gelatinsponge according to our usual protocol and were allowed to survive 1week. At time of surgery, rats were also implanted with osmoticmini-pumps subcutaneously with the delivery catheter placed in the brainat the site of injury. Animals received pumps with either glibenclamide(1 μM at 0.5 μl/hr×7 days) or diazoxide (10 μM at 0.5 μl/hr×7 days). Nosystemic toxicity was observed, neurological behavior was not impaired,and animals appeared healthy and were not febrile.

Cryosections of injured brains were examined for GFAP. In animalsreceiving glibenclamide, a well defined gliotic capsule was visualizedthat was sharply demarcated from surrounding brain, with the inner zoneappearing to be densely populated by GFAP-positive cells (FIG. 9A;gelatin sponge to the right). By contrast, animals receiving diazoxideshowed an expanded GFAP-positive response that extended farther from theforeign body, with an outer region that was poorly demarcated, and aninner zone that was loose and not compact (FIG. 9B; gelatin sponge tothe right).

Cryosections were also examined with the nuclear label, DAPI. Insections from glibenclamide-treated animals, most of the labeling wasattributable to GFAP-positive astrocytes. However, in sections fromdiazoxide-treated animals, DAPI labeling showed “sheets” of smallnucleated cells (dull spots in FIG. 10A). On inspection, these sheets ofcells appeared to be polymorphonuclear leukocytes (PMNs, neutrophils).This was confirmed by labeling with MMP-8, a PMN-specific marker (FIG.10B). It is important to note that no evidence of infection was present,and microbiological cultures of explanted materials showed no bacterialgrowth, including aerobic and anaerobic cultures, indicating that theinflammatory response was not due to infection.

Thus, protecting inner zone R1 astrocytes with glibenclamide appeared tohave restrained the overall GFAP-response to injury, whereas killinginner zone R1 astrocytes with diazoxide appeared to have caused anexpansion of the overall GFAP-response and recruitment of tremendousnumbers of neutrophils. These observations strongly reinforced theconcept of the “inner zone” of the gliotic capsule as being a uniqueentity, with a critical function in determining the overall response toinjury.

Example 8 SUR1 in Multiple Brain Pathologies

Tissues were obtained from the 3 rat models (trauma, abscess and stroke)and from human metastatic tumor, and double immunolabeling was performedwith antibodies directed against GFAP and SUR1. Low power views showed alayer of tissue adjacent to the gelatin sponge implant with positiveimmunolabeling for GFAP that coincided with positive immunolabeling forSUR1 (FIG. 11A,B). Examination of individual cells at high power showedthat the SUR1 immunolabel was present in large stellate-shapedastrocytes, confirming the presence of SUR1-positive R1 astrocytes inthe inner zone of the gliotic capsule surrounding a foreign body implant(FIG. 11C).

A brain abscess model in the rat was studied. The abscess was producedby implanting an autologous fecal pellet subcortically under generalanesthesia. These animals survived quite well, although they showedevidence of mild weight loss. When sacrificed 1 week after surgery, apurulent cavity was found surrounded by a gliotic capsule. Low powerviews of the gliotic capsule adjacent to the area of puss showed cellswith positive immunolabeling for GFAP that coincided with positiveimmunolabeling for SUR1 (FIG. 11D,E). Examination of individual cells athigh power showed that the SUR1 immunolabel was present in largestellate-shaped astrocytes, confirming the presence of SUR1-positive R1astrocytes in the inner zone of the gliotic capsule surrounding brainabscess (FIG. 11F).

A standard stoke model in the rat was studied. The stroke was producedby intra-carotid insertion of a thread up to the bifurcation of theinternal carotid artery, placed under general anesthesia. Animalssurviving the stroke were sacrificed at 1 week and the brain wasexamined Low power views of tissues adjacent to the area of strokeshowed cells with positive immunolabeling for GFAP that coincided withpositive immunolabeling for SUR1 (FIG. 11G,H). Examination of individualcells at high power showed that the SUR1 immunolabel was present inlarge stellate-shaped astrocytes, confirming the presence ofSUR1-positive R1 astrocytes in the gliotic capsule surrounding stroke(FIG. 11I).

Tissue was obtained from humans undergoing surgery for resection ofmetastatic brain tumors. At surgery, the gliotic capsule that surroundsthe metastasis is readily distinguished from the tumor itself and fromedematous white matter. Low power views of the gliotic capsule adjacentto the metastasis showed cells with positive immunolabeling for GFAPthat coincided with positive immunolabeling for SUR1 (FIG. 11J,K).Examination of individual cells at high power showed that the SUR1immunolabel was present in large stellate-shaped astrocytes withmultiple well-developed processes, confirming the presence ofSUR1-positive R1 astrocytes in the gliotic capsule surroundingmetastatic brain tumor in humans (FIG. 11L).

These data show for the first time SUR1 up-regulation in reactiveastrocytes at the site of formation of a gliotic capsule consistent withexpression of SUR1-regulated NC_(Ca-ATP) channels in R1 astrocytes. Thedata indicate that SUR1 expression in R1 astrocytes in the glioticcapsule was a common phenomenon in numerous pathological conditions thataffect the brain. These data highlight a unique opportunity tomanipulate R1 astrocytes of the inner zone selectively by exploitingpharmacological agents that act at SUR1 and that can therefore determinedeath or survival of these cells.

Overall, these observations strongly reinforced the concept of the“inner zone” of the gliotic capsule as being a unique entity, distinctfrom the remainder of the gliotic capsule.

Example 9 The NC_(Ca-ATP) Channel and Necrotic Death

NC_(Ca-ATP) channels were studied in a rodent model of stroke. In thepenumbra, SUR1 labeling was found in stellate-shaped cells (FIG. 12A)that were also GFAP-positive. In the middle of the stroke, stellatecells were absent, but SUR1 labeling was found in round cells exhibitinga bleb-like appearance (FIG. 12B,C) that were also GFAP-positive (notshown). The round cells with blebbing in situ resembled reactiveastrocytes in vitro undergoing necrotic death after exposure to Naazide. The effect of glibenclamide vs. saline was determined.Glibenclamide or saline was administered via subcutaneously-implantedosmotic mini-pump (1 μM at 0.5 μl/hr). In saline treated rats, 3-daymortality after stroke was 68%, whereas in glibenclamide-treated rats,3-day mortality was reduced to 28% (n=29 in each group; p<0.001, by χ²).In separate animals, the stroke hemisphere in glibenclamide-treated ratscontained only half as much excess water as in saline-treated rats (n=5in each group; p<0.01, by t-test), confirming an important role of theNC_(Ca-ATP) channel in edema formation.

SUR1 was also studied in a rodent model of trauma. The effect of directinfusion of drugs into the site of trauma was examined using animplanted osmotic mini-pump. The channel inhibitor, glibenclamide, wasused to reduce death of reactive astrocytes, and the channel activator,diazoxide, to promote astrocyte death. Glibenclamide infusion reducedthe overall injury response, stabilized the gliotic capsule around theforeign body implant, and minimized the inflammatory response comparedto control.

Conversely, diazoxide essentially destroyed the gliotic capsule andincited a huge inflammatory response, characterized by massive influx ofpolymorphonuclear cells (PMNs) (FIG. 10A, B). These data suggested thatNC_(Ca-ATP) channel plays a critical role in the injury response, andthey strongly support the hypothesis that inflammation is closely linkedto activity of the NC_(Ca-ATP) channel and necrotic death of reactiveastrocytes.

Example 10 Permanent MCA Models

Adult male or female Wistar rats (275-350 gm) were fasted overnight thenanesthetized (Ketamine, 60 mg/kg plus Xylazine, 7.5 mg/kg, i.p.). Theright femoral artery was cannulated, and physiological parameters,including temperature, pH, pO₂, pCO₂ and glucose were monitored. Using aventral cervical incision, the right external carotid andpterygopalatine arteries were ligated. The common carotid artery wasligated proximally and catheterized to allow embolization of theinternal carotid artery.

For thromboembolic (TE) stroke, 7-8 allogeneic clots, 1.5 mm long, wereembolized. Allogeneic, thrombin-induced, fibrin-rich blood clots wereprepared (Toomy et al., 2002).

For large MCA strokes with malignant cerebral edema (MCE), the inventorsfirst embolized microparticles (Nakabayashi et al., 1997) [polyvinylalcohol (PVA) particles; Target Therapeutics, Fremont Calif.; 150-250 μmdiameter, 600 μg in 1.5 ml heparinized-saline], followed by standardpermanent intraluminal suture occlusion (Kawamura et al., 1991) using amonofilament suture (4-0 nylon, rounded at the tip and coated withpoly-L-lysine) advanced up to the ICA bifurcation and secured in placewith a ligature.

After stroke, animals are given 10 ml glucose-free normal saline bydermoclysis. Rectal temperature was maintained at ≈37° C. using aservo-controlled warming blanket until animals awoke from anesthesia.Blood gases and serum glucose at the time of stroke were: pO₂, 94±5 mmHg; pCO₂, 36±5 mm Hg; pH, 7.33±0.01; glucose 142±6 mg/dl in controls andpO₂, 93±3 mm Hg; pCO₂, 38±2 mm Hg; pH, 7.34±0.01; glucose 152±7 mg/dl inglibenclamide-treated animals.

With both models, animals awoke promptly from anesthesia and movedabout, generally exhibited abnormal neurological function, typicallycircling behavior and hemiparesis. Mortality with the thromboembolic(TE) model was minimal, whereas with the malignant cerebral edema (MCE)model, animals exhibited delayed deterioration, often leading to death.Most deaths occurred 12-24 hr after MCA occlusion, with necropsiesconfirming that death was due to bland infarcts. Rarely, an animal died<6 hr after stroke and was found at necropsy to have a subarachnoidhemorrhage, in which case it was excluded from the study. Mortality inuntreated animals with MCE and bland infarcts was 65%, similar to thatin humans with large MCA strokes (Ayata & Ropper, 2002).

Example 11 Studies on Stroke Size, Mortality, Tissue-Water, and DrugLocalization

After MCA occlusion (both TE and MCE models), mini-osmotic pumps (Alzet2002, Durect Corporation, Cupertino, Calif.) were implantedsubcutaneously that delivered either saline or glibenclamide (Sigma, St.Louis, Mo.; 300 μM or 148 μg/ml, 0.5 μl/hr subcutaneously, no loadingdose). Stroke size (TE model), measured as the volume of TTC(−) tissuein consecutive 2 mm thick slices and expressed as the percent ofhemisphere volume, was compared 48 after stroke in 2 treatment groups,each comprised of 10 male rats, treated with either saline orglibenclamide. Mortality (MCE model) was compared during the first weekafter stroke in 2 treatment groups, each comprised of 29 rats (19 femaleplus 10 male), treated with either saline or glibenclamide. Edema (MCEmodel) was compared at 8 hr after stroke in 2 treatment groups, eachcomprised of 11 male rats, treated with either saline or glibenclamide;rats in each of these 2 treatment groups were subdivided into 2subgroups, with the first of these being used to analyze water in theentire involved hemisphere (no TTC processing), and the second beingused to analyze water in the TTC(+) vs. TTC(−) portions of the involvedhemisphere. For localization of fluorescent-tagged drug, 20 male ratswere subjected to MCA stroke (MCE model) and were implanted withmini-osmotic pumps that delivered BODIPY-conjugated glibenclamide(BODIPY-FL-glyburide, Molecular Probes, Eugene, Oreg.; 300 μM or 235μg/ml, 0.5 μl/hr subcutaneously, no loading dose). Of these, 15 ratswere used for validation of drug action (mortality, tissue water andglucose) and 5 rats were used for determination of drug distribution.

Example 12 Immunolabeling

Brains were perfusion-fixed (4% paraformaldehyde) and cryoprotected (30%sucrose). Cryosections (10 μm) were prepared and immunolabeled usingstandard techniques (Chen et al., 2003). After permeabilizing (0.3%Triton X-100 for 10 min), sections were blocked (2% donkey serum for 1hr; Sigma D-9663), then incubated with primary antibody directed againstSUR1 (1:300; 1 hr at room temperature then 48 h at 4° C.; SC-5789; SantaCruz Biotechnology). After washing, sections were incubated withfluorescent secondary antibody (1:400; donkey anti-goat Alexa Fluor 555;Molecular Probes, Oreg.). For co-labeling, primary antibodies directedagainst NeuN (1:100; MAB377; Chemicon, Calif.); GFAP (1:500; CY3conjugated; C-9205; Sigma, St. Louis, Mo.) and vWf (1:200; F3520, Sigma)were used and tissues were processed according to manufacturers'recommendations. Species-appropriate fluorescent secondary antibodieswere used as needed. Fluorescent signals were visualized usingepifluorescence microscopy (Nikon Eclipse E1000).

Example 13 TTC Staining, Stroke Size

Freshly harvested brains were cut into 2-mm thick coronal sections, andslices were exposed to TTC (0.125% w/v in 62.5 mM Tris-HCl, 13 mM MgCl₂,1.5% dimethylformamide) for 30 min at 37° C. For stroke size, stainedsections were photographed and images were analyzed (Scion Image) todetermine the percent of the involved hemisphere occupied by TTC(−)tissue; no correction for edema was performed. For some determinationsof water or SUR1 protein content, individual coronal sections weredivided under magnification into 3 parts: (i) the non-involved, controlhemisphere; (ii) the TTC(+) portion of the involved hemisphere; (iii)the TTC(−) portion of the involved hemisphere. For each animal, pooledtissues from the 3 parts were then processed for tissue watermeasurements or for Western blots.

Example 14 Tissue Water Content

Tissue water was quantified by the wet/dry weight method (Hua et al.,2003). Tissue samples were blotted to remove small quantities ofadsorbed fluid. Samples were weighed with a precision scale to obtainthe wet weight (WW), dried to constant weight at 80° C. and low vacuum,and then reweighed to obtain the dry weight (WD). The percent H₂O ofeach tissue sample was then calculated as (WW−WD)×100/WW.

Example 15 Immunoblots

Tissues lysates and gels were prepared (Perillan et al., 2002).Membranes were developed for SUR1 (SC-5789; Santa Cruz Biotechnology),Kir6.1 (Santa Cruz) or Kir6.2 (Santa Cruz). Membranes were stripped andre-blotted for β-actin (1:5000; Sigma, St. Louis, Mo.), which was usedto normalize the primary data. Detection was carried out using the ECLsystem (Amersham Biosciences, Inc.) with routine imaging andquantification (Fuji LAS-3000).

Example 16 In Situ Hybridization

Non-radioactive digoxigenin-labeled probes were made according to themanufacturer's protocol (Roche) using SP6 or T7 RNA polymerase. RNAdig-labeled probes (sense and anti-sense) were generated from pGEM-Teasy plasmids (Promega) with the SUR1 insert (613 bp) flanked by theprimers: 5′ AAGCACGTCAACGCCCT 3′ (SEQ ID NO: 1) (forward); 5′GAAGCTTTTCCGGCTTGTC 3′ (SEQ ID NO: 2) (reverse). Fresh-frozen (10 μm) orparaffin-embedded (4 μm) sections of rat brain (3, 6, 8 hours after MCAstroke) were used for in situ hybridization (Anisimov et al., 2002).

Example 17 Inner Zone of the Gliotic Capsule

To assess if other causes of hypoxia, for example arterial occlusion,resulted in up-regulation of SUR1, two rodent models of permanent focalcerebral ischemia described in Example 10 were used.

The MCE model was used to evaluate SUR1 protein and mRNA, and to assesseffects of SUR1 inhibition on edema and survival, while the TE model wasused to measure effects of SUR1 inhibition on stroke size. Absence ofperfusion (FIG. 14A), TTC staining (Mathews et al., 2000) (FIG. 14B) andGFAP immunolabeling were used to distinguish infarct from peri-infarctregions.

SUR1 expression increased transiently in the core of the infarct. Here,an increase in SUR1 became evident as early as 2-3 hr after MCAocclusion (FIG. 14D), well before onset of necrosis, and laterdisappeared as necrosis set in (FIG. 14C, right side of figure). Atthese early times before necrosis, SUR1 was very prominent in neuronsthat co-labeled with NeuN (FIG. 14D-F).

In peri-infarct regions, including the classical ischemic “watershed”zone between anterior cerebral artery (ACA) and MCA territories, SUR1expression increased later than in the core but was sustained. By 6-12hr, SUR1 expression sharply demarcated infarct and peri-infarct areas(FIG. 14C). Here, SUR1 expression was found in neurons, astrocytes andcapillary endothelial cells, as shown by co-labeling with NeuN, GFAP(FIG. 14G-I) and von Willebrand factor (FIG. 14J-L), respectively. SUR1is not normally expressed in such abundance in these cortical andsubcortical areas (Treherne & Ashford, 1991; Karschin et al., 1997) asis evident in contralateral tissues (FIG. 14C, left side of figure).

Western blots showed an increase in expression of SUR1 protein, mostprominently in peri-infarct regions (FIG. 15A-D). However, thepore-forming subunits of K_(ATP) channels, Kir6.1 or Kir6.2, were notup-regulated (FIG. 15C-D). In situ hybridization showed SUR1 transcriptsin neurons and capillaries from regions of ischemia that were notpresent in control tissues (FIG. 15E-G), suggesting that SUR1, but notK_(ATP) channels, was transcriptionally up-regulated in cerebralischemia.

Thus, these data suggest that SUR1, but not Kir6.1 or Kir6.2, istranscriptionally up-regulated in cerebral ischemia, first in regionsthat are destined to undergo necrosis, and later in peri-infarctregions.

Example 18 SUR1 Up-Regulation

FIG. 15A-G of the showed that SUR1 was significantly up-regulated instroke. It also showed that the pore-forming subunits, Kir6.1 andKir6.2, were not up-regulated in stroke, suggesting that K_(ATP)channels were not involved. To prove that SUR1 up-regulation is due toNC_(Ca-ATP) channels and not to K_(ATP) channels, patch clamp recordingsof neurons and endothelial cells from ischemic regions were performed.Large neuron-like cells were enxymatically isolated 3-hr (FIG. 16A) and6-hr after stroke. Patch clamp study was carried out using Cs⁺ in thebath and pipette, to block all K⁺ channels including K_(ATP) channels.These experiments showed robust cation channel activity that was blockedby glibenclamide, as predicted for the NC_(Ca-ATP) channel (FIG. 16B).In addition, when channel activity was recorded with K⁺, the slopeconductance was 34 pS (FIG. 16C,D), as previously reported in freshlyisolated R1 astrocytes, and much less than the 70-75 pS reported forK_(ATP) channels.

Example 19 Function of SUR1 in Cerebral Ischemia

To determine the function of SUR1 that was newly expressed in cerebralischemia, the effects of glibenclamide, a highly selective inhibitor ofSUR1 was studied. The effect of glibenclamide on mortality (MCE model)was studied. In a large group of animals, both male and female,treatment with glibenclamide resulted in a dramatic reduction inmortality compared to saline, from 65% to 24% (p<0.002; FIG. 17A).

Since glibenclamide had been shown to ameliorate cytotoxic edema ofastrocytes induced by energy depletion (Chen et al., 2003), it wasreasoned that the beneficial effect on mortality was related to edema.The effect of glibenclamide on the formation of edema 8 hr afterinduction of stroke (MCE model) was examined This is a time thatpreceded death of any animal in the mortality study. In the first of twoexperiments, water content in the involved and uninvolved hemisphereswas measured using the methods described in Example 14. For the controlhemisphere, water was 77.9±0.2%. For the involved hemisphere, water roseby 3.4%, to 81.3±0.5% for the group treated with saline, whereas it roseby only 2.0%, to 79.9±0.3%, for the group treated with glibenclamide.These values were significantly different (p<0.05), consistent with animportant role of SUR1 in formation of edema.

Next, to better characterize the location of edema, the water contentafter dividing coronal brain sections into viable TTC(+) and non-viableTTC(−) parts was examined Water in the uninvolved hemisphere was78.0±0.1% (FIG. 17B), similar to the previous value of 77.9±0.2%,indicating that TTC processing had not altered water content. For theinvolved hemisphere, water in the TTC(+) tissue rose by 5.4%, to83.4±1.1% for the group treated with saline, whereas it rose by only2.5%, to 80.5±0.3%, for the group treated with glibenclamide (FIG. 17B).These values were significantly different (p<0.05). By contrast, valuesfor water in TTC(−) tissues, 78.7±1.0% and 78.6±0.4% with saline andwith glibenclamide, respectively, were not different (p=0.97), and wereonly slightly higher than the value for the uninvolved hemisphere(78.0%), reflecting a need for ongoing blood flow to increase tissuewater (FIG. 17B) (Ayata & Ropper, 2002).

In these animals, serum glucose at 8 hr when edema was measured remainedin a range unlikely to have an effect on ischemia-induced damage (Li etal., 1994; Wass & Lanier, 1996) (122±4 vs. 93±3 mg/dl for saline andglibenclamide-treated animals, respectively; 11 rats/group). Together,these data indicated that the edema was located almost entirely inviable peri-infarct (penumbral) tissue adjacent to the early core of thestroke, and that glibenclamide was highly effective in reducing it,consistent with an important role for SUR1 in formation of edema.

Thus, the data with low-dose glibenclamide, which is highly selectivefor SUR1 (Gribble & Reimann, 2003; Meyer et al., 1999) providedcompelling evidence of a critical role for SUR1 in formation of cerebraledema.

Example 20 The Effect of Stroke Size

A non-lethal thromboembolic (TE) model was used to assess stroke size 48hr after induction of stroke.

With the TE model, glibenclamide treatment resulted in a highlysignificant reduction in stroke volume, compared to saline controls(32.5±4.9% vs. 15.5±2.3%; p<0.01) (FIG. 17C-E). Essentially all animals,regardless of treatment group, suffered infarctions involving the basalganglia, which were supplied by terminal lenticulostriate arterioles.However, reduced stroke volumes in the glibenclamide group were oftenassociated with marked sparing of the cerebral cortex (FIG. 17C-D), aphenomenon previously reported with decompressive craniectomy (Doerfleret al., 2001). With glibenclamide, cortical sparing may reflect improvedleptomeningeal collateral blood flow due to reduced cerebral edema andreduced intracranial pressure.

Example 21 MCE Model Following Stroke

The fluorescent derivative, BODIPY-glibenclamide, was used to labeltissues in vivo following stroke (MCE model).

When delivered in the same manner as the parent compound, thefluorescent derivative exhibited similar protective effects, but wasless potent [7-day mortality, 40% (n=10); water in the TTC(+) portion ofthe involved hemisphere at 8 hr, 82.7±1.4% (n=5); serum glucose, 109±4mg/dl], consistent with reduced efficacy of the labeled drug (Zunkler etal., 2004). The low systemic dose of drug used yielded minimal labelingin the uninvolved hemisphere (FIG. 18B) and pancreas, and none in theunperfused core of the stroke. However, cells in peri-infarct regionswere clearly labeled, with well-defined labeling of large neuron-likecells and of microvessels (FIG. 18A), including capillaries (FIG. 18C),that showed prominent expression of SUR1 (FIG. 18D). Preferentialcellular labeling in ischemic brain likely reflected not only anincrease in glibenclamide binding sites, but also an increase in uptake,possibly due to alteration of the blood brain barrier.

Thus, the data indicated the presence of NC_(Ca-ATP) channels incapillary endothelium and neurons in addition to their previouslydescribed presence in astrocytes (Chen et al., 2001; Chen et al., 2003).Additional patch clamp experiments on neurons and microvessels isolatedfrom ischemic cortex 1-6 hr after MCA occlusion (MCE model) confirmedthe presence of NC_(Ca-ATP) channels, showing a non-selective cationchannel of around 30-35 pS conductance, that was easily recorded withCs⁺ as the charge carrier, and that was blocked by glibenclamide. Thischannel was not present in cells from non-ischemic cerebral tissues.

In view of the above, it is suggested that SUR1-regulated NC_(Ca-ATP)channels that are opened by ATP depletion and that are newly expressedin ischemic neurons, astrocytes and endothelial cells constitute animportant, heretofore unidentified pathway for Na⁺ flux required forformation of cytotoxic and ionic edema. Together, these findings suggesta critical involvement of SUR1 in a new pathway that determinesformation of edema following cerebral ischemia. Molecular therapiesdirected at SUR1 may provide important new avenues for treatment of manytypes of CNS injuries associated with ischemia.

Example 22 Co-Administration of Glibenclamide and tPA

A rodent model of thromboembolic stroke was used (Aoki et al., 2002;Kijkhuizen et al., 2001; Kano et al., 2000; Sumii et al., 2002; Tejimaet al., 2001). Briefly, male spontaneously hypertensive rats that havebeen fasted overnight are anesthetized using halothane (1-1.5% in a70/30 mixture of N₂O/O₂) with spontaneous respiration (Lee et al., 2004;Sumii et al., 2002). Rectal temperature was maintained at ≈37° C. with athermostat-controlled heating pad. The right femoral artery wascannulated, and physiological parameters, including temperature, meanblood pressure, pH, pO₂, and pCO₂, glucose were monitored. Temporaryfocal ischemia was obtained with an embolic model that used allogeneicclots to occlude the MCA. Allogeneic, thrombin-induced, fibrin-richblood clots were prepared using methods adapted from Niessen et al.(Asahi et al., 2000; Niessen et al., 2003; Sumii et al., 2002). Sevenclots, 1.5 mm long, were used for embolizing.

Using a ventral cervical incision, the internal and external carotidarteries were exposed. The external carotid artery and pterygopalatinearteries were ligated. Removable surgical clips were applied to thecommon and internal carotid arteries. The modified PE-50 cathetercontaining the clots was inserted retrograde into the external carotidartery and advanced up to the internal carotid artery. The temporaryclips were removed, and the clots were injected. Incisions were closed.

After stroke, animals were given glucose-free normal saline, 10 mltotal, by dermoclysis. Temperature was maintained until animals wereawake and were moving about.

Just prior to the time designated for treatment (reperfusion), animalswere re-anesthetized and the femoral vein was cannulated. At the timedesignated for treatment, saline, or a loading dose of glibenclamide(1.5 μg/kg, i.v., Sigma, St. Louis) was first administered. Then,reperfusion was achieved with i.v. administration of rtPA (10 mg/kg,Alteplase, Genetech; dissolved in 2 ml distilled water, given over 30min) (Buesseb et al., 2002). Then, using a dorsal thoracic incision, amini-osmotic pump (Alzet 2002, Durect Corporation, Cupertino, Calif.)was implanted subcutaneously that delivered either saline orglibenclamide (300 μM or 148 μg/ml, 0.5 μl/hr s.q.). Physiologicalparameters, including temperature, mean blood pressure (tail cuffplethysmography), blood gases and glucose were monitored.

At the same time of 6 hr, animals were co-treated with either saline orglibenclamide (loading dose of 1.5 μg/kg i.v. plus implantation of amini-osmotic pump containing 148 μg/m1=300 μM delivered at ½ μl/hr).Animals were euthanized 24 hr following stroke and brains were perfusedto remove blood from the intravascular compartment. Coronal sections ofthe fresh brains were prepared and photographed, following whichsections were processed for TTC staining to identify areas ofinfarction.

All animals (5/5) co-treated with saline showed large regions ofhemorrhagic conversion in cortical and subcortical parenchymal areas ofinfarction, along with evidence of intraventricular hemorrhage (FIG.19A-D). In contrast, only 1/5 animals co-treated with glibenclamide hadhemorrhagic conversion, with 4/5 showing no evidence of hemorrhage (FIG.19E-H).

These data suggest that there was protection from hemorrhagic conversionwith the administration of glibenclamide, as well as reduction in strokesize, ionic edema, and vasogenic edema.

Example 23 Isolation of Brain Capillaries and Endothelial Cells

The method was adapted in part from Harder et al. (1994) withmodifications as previously reported (Seidel, 1991). Briefly, a rat wasdeeply anesthetized, the descending aorta was ligated, the right atriumwas opened and the left ventricle was cannulated to allow perfusion of50 ml of a physiological solution containing a 1% suspension of ironoxide particles (particle size, 10 μm; Aldrich Chemical Co.). The brainwas removed, the pia and pial vessels were stripped away and thecortical mantel is minced into pieces 1-2 mm³ with razor blades. Thetissue pieces were incubated with trypsin plus DNAse and then sievedthrough nylon mesh (210 μm). Retained microvessels were resuspended incollagenase, agitated and incubated at 37° C. for an additional 10 min.To terminate the digestion, microvessels were adhered to the side of thecontainer with a magnet and washed repeatedly to remove enzyme andcellular debris.

Using these methods yielded healthy-appearing microvascular structuresthat were suitable for further digestion to obtain single cells (FIG.21) for further experiments.

Isolated endothelial cells were studied using freshly isolatedendothelial cells using a nystatin-perforated patch technique. Withphysiological solutions, the cells exhibited a prominent, stronglyrectifying inward current at negative potentials, and a modest outwardcurrent at positive potentials (FIG. 22A), yielding a characteristiccurrent-voltage curve with near-zero current at intermediate potentials(FIG. 22C), similar to previous observations in freshly isolatedendothelial cells (Hogg et al., 2002). When K⁺ in the pipette solutionwas replaced with Cs⁺, K⁺ channel currents were completely blocked. Inendothelial cells, this yielded a current-voltage curve that was linear(FIG. 22E). These data demonstrated that voltage dependent channels infreshly isolated endothelial cells are exclusively K+channels that donot carry Na⁺.

Example 24 Isolation of Neurons

Neurons were isolated from vibratome sections Immunolabeling experimentsindicated that ischemic NeuN-positive neurons expressed SUR1 within 2-3hr after MCAO, before necrosis was evident. Therefore, tissues wereprepared at 2-3 hr after MCAO. The brain was divided coronally at thelevel of the bregma, and cryosections were prepared from one half andvibratome sections were prepared from the other half. Cryosections (10μm) were used for TTC staining (Mathews et al., 2000) or alternatively,high-contrast silver infarct staining (SIS), (Vogel et al., 1999) toidentify the region of ischemia, and for immunolabeling, to verify SUR1up-regulation in neurons double labeled for NeuN. Vibratome sections(300 μm) were processed (Hainsworth et al., 2000; Kay et al., 1986;Moyer et al., 1998) to obtain single neurons for patch clamping.Selected portions of coronal slices were incubated at 35° C. in HBSSbubbled with air. After at least 30 min, the pieces were transferred toHBSS containing 1.5 mg/ml protease XIV (Sigma). After 30-40 min ofprotease treatment, the pieces were rinsed in enzyme-free HBSS andmechanically triturated. For controls, cells from mirror-image corticalareas in the uninvolved hemisphere were used. Cells were allowed tosettle in HBSS for 10-12 min in a plastic Petri dish mounted on thestage of an inverted microscope. Large and medium-sized pyramidal-shapedneurons were selected for recordings. At this early time of 2-3 hr, onlyneurons and capillaries, not astrocytes, show up-regulation of SUR1.

Once the cells were isolated patch clamp experiments using well knownmethods including whole-cell, inside-out, outside-out and perforatedpatch were used (Chen et al., 2003; Chen et al., 2001; Perillan et al.,2002; Perillan et al., 2000; Perillan et al., 1999)

Example 25 MMP Inhibition by Glibenclamide

Activation of MMP-9 & MMP-2 in stroke tissue was compared to controls.Briefly, gelatinase activity of recombinant enzyme and stroke tissueunder control conditions (CTR), in presence of glibenclamide (10 μM),and in presence of MMP-inhibitor II (300 nM; Calbiochem).

Next, the supernatants underwent a gelatinase purification process withgelatin-Sepharose 4B (Pharmacia), and Zymography was performed on thepurified supernatants in sodium dodecyl sulfate gels containing gelatin(Rosenberg, 1994). Dried gels were scanned with a transparency scanner,and images were analyzed by densitometry. The relative lysis of anindividual sample was expressed as the integrated density value of itsband and divided by the protein content of the sample.

Zymography confirmed that gelatinase activity was increased after stroke(FIG. 20A), and showed that gelatinase activity assayed in the presenceof glibenclamide (FIG. 20B, Glibenclamide) was the same as that assayedwithout (FIG. 20B, CTR), although gelatinase activity was stronglyinhibited by commercially available MMP inhibitor II (FIG. 20B,MMP-2/MMP-9 inhibitor). These data demonstrated that glibenclamide didnot directly inhibit gelatinase activity, and suggested that thereduction of hemorrhagic conversion observed with glibenclamide likelycame about due to a beneficial, protective effect of glibenclamide onischemic endothelial cells.

Example 26 Up-Regulation of SUR1-mRNA in Stroke

Additional molecular evidence for involvement of SUR1 in stroke wasobtained using quantitative RT-PCR.

Total RNA was extracted and purified from samples of homogenized braintissues contralateral (CTR) and ipsilateral to MCAO (STROKE) usingguanidine isothyocyonatye. cDNA was synthesized with 4 μg of total RNAper 50 μl of reaction mixture using TaqMan RT kit (Applied Biosystems).Relative values of SUR1-mRNA were obtained by normalizing to H1f0(histone 1 member 0). The following probes were used SUR1 forward:GAGTCGGACTTCTCGCCCT (SEQ ID NO: 3); SUR1 reverse: CCTTGACAGTGGCCGAACC(SEQ ID NO: 4); SUR1 TaqMan Probe: 6-FAM-TTCCACATCCTGGTCACACCGCTGT (SEQID NO: 5) TAMRA; H1f0 forward: CGGACCACCCCAAGTATTCA (SEQ ID NO: 6); H1f0reverse: GCCGGCACGGTTCTTCT (SEQ ID NO: 7); H1f0 TaqMan Probe:6-FAM-CATGATCGTGGCTGCTA TCCAGGCA(SEQ ID NO: 8)-TAMRA.

These data showed that mRNA for SUR1 was significantly increased in thecore region, 3 hr after MCAO (FIG. 23).

Example 27 SUR1 Knockdown (SUR1KD) is Protective

To further test involvement of SUR1, SUR1 expression was “knocked down”in situ by infusing oligodeoxynucleotide (ODN) for 14 days using amini-osmotic pump, with the delivery catheter placed in the gelfoamimplantation site in the brain, in the otherwise standard model we usefor R1 astrocyte isolation (Perillan et al., 1980, Perillan et al.,2002, Perillan et al., 2000, Perillan et al., 1999). Knockdown of SUR1expression (SUR1KD) was achieved using antisense (AS;5′-GGCCGAGTGGTTCTCGGT-3′) (SEQ ID NO: 9) (Yokoshiki et al., 1999)oligodeoxynucleotide (ODN), with scrambled (SCR;5′-TGCCTGAGGCGTGGCTGT-3′ (SEQ ID NO: 10)) ODN being used as control.

Immunoblots of gliotic capsule showed significant reduction in SUR1expression in SUR1 knockdown (SUR1KD) tissues compared to controlsreceiving scrambled sequence ODN (FIGS. 24A and 24B).

The inventors enzymatically isolated single cells from SUR1KD andcontrols using a standard cell isolation protocols described above (Chenet al., 2003) to assess functional responses to ATP depletion induced byNa azide. In R1 astrocytes from control tissues, Na azide (1 mM) causedrapid depolarization due to Na⁺ influx attributable to activation ofNC_(Ca-ATP) channels (FIG. 24C). Notably, this depolarizing response wasopposite the hyperpolarizing response observed when K_(ATP) channelswere activated. In R1 astrocytes from SUR1KD, however, Na azide hadlittle effect on resting membrane potential (FIG. 24D). In controls,application of Na azide resulted in depolarization of 64±3.7 mV, whereasin cells for SUR1KD, depolarization was only 8.7±1.7 mV (FIG. 24E).

In addition, membrane blebbing that typically follows exposure to Naazide was not observed in cells from SUR1KD, confirming the role forSUR1 in cytotoxic edema of R1 astrocytes.

Example 28 Molecular Factors that Regulate SUR1 Expression

Based on work in pancreatic β cells, a number of SP1 transcriptionfactor binding sites have been identified in the proximal SUR1 promoterregion that are considered to be important for activation of SUR1transcriptional activity (Ashfield et al., 1998; Hilali et al., 2004).Notably, SP1 has essentially not been studied in stroke (Salminen etal., 1995).

Briefly, the ischemic peri-infarct tissues was immunolabeled for SP1,which is important for SUR1 expression, for HIF1α, which is widelyrecognized to be up-regulated in cerebral ischemia (Semenza 2001; Sharpet al., 2000) and for SUR1 itself. SP1 was prominently expressed inlarge neuron-like cells and in capillaries (FIG. 25A, 25C) in regionsconfirmed to be ischemic by virtue of expression of HIF1α (FIG. 25B).Notably, capillaries that expressed SP1 also showed prominent expressionof SUR1 (FIG. 25C, 25D). Contralateral control tissues showed littleimmunolabeling for SP1 and none for HIF1α (FIG. 25E, 25F).

Nuclear SP1 localization was significantly augmented early-on in stroke(FIG. 26A, 26B), and nuclear SP1 was found in large neuron-like cellsthat express SUR1 following MCAO (FIG. 26C).

HIF1α knock-down animals were obtained by infusion of antisenseoligodeoxynucleotide at the site of gelfoam implant. FIG. 27 confirmsthe HIF1α knock-down animals results in a significant decrease in SUR1expression (FIG. 27B, 27D), providing strong evidence that not only SP1but also HIF1α is likely to be an important regulator of SUR1expression.

REFERENCES

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

-   Aguilar-Bryan L, et al., Science. 1995; 268:423-426.-   Ammala C, et al., Nature. 1996; 379:545-548.-   Anisimov, S. V., et al., Mech. Dev. 117, 25-74 (2002).-   Aoki K, et al., Acta Neuropathol (Berl). 2003; 106:121-124.-   Arteel G E, et al., Eur J Biochem. 1998; 253:743-750.-   Ashcroft F M. Science. 1998; 282:1059-1060.-   Ayata, C. & Ropper, A. H. J. Clin. Neurosci. 9, 113-124 (2002).-   Babenko A P, et al., Annu Rev Physiol. 1998; 60:667-687.-   Ballanyi, K. J. Exp. Biol. 207, 3201-3212 (2004).-   Barclay J, et al., J Neurosci. 2002; 22:8139-8147.-   Baukrowitz T, et al., Science. 1998; 282:1141-1144.-   Becker J B, et al., Ann N Y Acad Sci. 2001; 937:172-187.-   Beyer C, et al., J Steroid Biochem Mol Biol. 2002; 81:319-325.-   Blurton-Jones M, et al., J Comp Neurol. 2001; 433:115-123.-   Bussink J, et al., Radiat Res. 2000; 154:547-555.-   Cevolani D, et al., Brain Res Bull. 2001; 54:353-361.-   Chen M, et al., J Neurosci. 2003; 23:8568-8577.-   Chen M, Simard J M. J Neurosci. 2001; 21:6512-6521-   Chen H., et al., J. Neurol. Sci. 118, 109-6 (1993).-   Choi I, et al., Mol Cell Endocrinol. 2001; 181:139-150.-   Cress A E. Biotechniques. 2000; 29:776-781.-   Dalton S, et al., Glia. 2003; 42:325-339.-   Dhandapani K, et al., Endocrine. 2003; 21:59-66.-   Dhandapani K M, et al., Biol Reprod. 2002; 67:1379-1385.-   Dhandapani K M, et al., BMC Neurosci. 2002; 3:6.-   Diab A, et al., Infect Immun 1999; 67:2590-2601.-   Doerfler, A., et al., Stroke 32, 2675-2681 (2001).-   Drain P, et al., Proc Natl Acad Sci USA. 1998; 95:13953-13958.-   Dubik D et al., Oncogene. 1992; 7:1587-1594.-   El Ashry D, et al., J Steroid Biochem Mol Biol. 1996; 59:261-269.-   Enkvetchakul D, et al., Biophys J. 2000; 78:2334-2348.-   Falk E M, et al., Pharmacol Biochem Behav. 2002; 72:617-622.-   Fischer S, et al., J Cell Physiol. 2004; 198:359-369.-   Foy M R, et al., Brain Res. 1984; 321:311-314.-   Fujita A, et al., Pharmacol Ther. 2000; 85:39-53.-   Garcia-Estrada J, et al., Brain Res. 1993; 628:271-278.-   Garcia-Ovejero D, et al., J Comp Neurol. 2002; 450:256-271.-   Garcia-Segura L M, et al., Frog Neurobiol. 2001; 63:29-60.-   Garlid K D, et al., Circ Res. 1997; 81:1072-1082.-   Giaccia A J, et al., Int J Radiat Oncol Biol Phys. 1992; 23:891-897.-   Gribble, F. M. & Reimann, F Diabetologia 46, 875-891 (2003).-   Grover G J. Can J Physiol Pharmacol. 1997; 75:309-315.-   Guo X Z, et al., Cell Res. 2001; 11:321-324.-   Hainsworth et al., Neuropharmacology. 2001; 40:784-791.-   Hale L P, et al., Am J Physiol Heart Circ Physiol. 2002;    282:H1467-H1477.-   Halstead J, et al., J Biol Chem. 1995; 270:13600-13603.-   Harder et al., Am J Physiol. 1994; 266:H2098-H2107.Haruna T, et al.,    Pflugers Arch. 2000; 441:200-207.-   Haug A, et al., Arch Toxicol. 1994; 68:1-7.-   Higashijima T, et al., J Biol Chem. 1990; 265:14176-14186.-   Higgins C F. Annu Rev Cell Biol. 1992; 8:67-113.-   Hiroi H, et al., J Mol Endocrinol. 1999; 22:37-44.-   Hobbs M V, et al., J Immunol. 1993; 150:3602-3614.-   Hogg et al., FEBS Lett. 2002; 522:125-129.-   Hogg et al., Lung. 2002; 180:203-214.-   Hohenegger M, et al., Proc Natl Acad Sci U S A. 1998; 95:346-351.-   Honda K, et al., J Neurosci Res. 2000; 60:321-327.-   Hossain M A, et al., J Biol Chem. 2000; 275:27874-27882.-   Hua Y, et al., J Cereb Blood Flow Metab. 2003; 23:1448-1454.-   Hunt R A, et al., Hypertension. 1999; 34:603-608.-   Huovinen R, et al., Int J Cancer. 1993; 55:685-691.-   Ignotz R A, et al., J Cell Biochem. 2000; 78:588-594.-   Inagaki N, et al., Neuron. 1996; 16:1011-1017.-   Isomoto S, et al., J Biol Chem. 1996; 271:24321-24324.-   Jain, Sci. Amer. 271: 58-65, 1994.-   Jorgensen M B, et al., Exp Neurol. 1993; 120:70-88.-   Jovanovic A, et al., Lab Invest. 1998; 78:1101-1107.-   Kakinuma Y, et al., Clin Sci (Lond). 2002; 103 Suppl 48:210S-214S.-   Kangas L. Cancer Chemother Pharmacol. 1990; 27:8-12.-   Kangas L. J Steroid Biochem. 1990; 36:191-195.-   Kanthasamy A, et al., Neuroscience. 2002; 114:917-924.-   Karschin, C., et al., FEBS Lett. 401, 59-64 (1997).-   Kawamura, S., et al., Acta Neurochir. (Wien.) 109, 126-132 (1991).-   Kay et al., J Neurosci Methods. 1986; 16:227-238.-   Ke C, et al., Neurosci Lett. 2001; 301:21-24.-   Kelly M J, et al., Steroids. 1999; 64:64-75.-   Kennedy A S, et al., Int J Radiat Oncol Biol Phys. 1997; 37:897-905.-   Kielian T, et al., J Immunol. 2001; 166:4634-4643.-   Kimura D. Sci Am. 1992; 267:118-125.-   Kohshi K, J Neurol Sci. 2003; 209:115-117.-   Koster J C, J Gen Physiol. 1999; 114:203-213.-   Kucich U, et al., Arch Biochem Biophys. 2000; 374:313-324.-   Kuiper G G, et al., Endocrinology. 1997; 138:863-870.-   Kuiper G G, et al., Proc Natl Acad Sci U S A. 1996; 93:5925-5930.-   Larsson O, et al., Diabetes. 2000; 49:1409-1412.-   Lawson K. Kidney Int. 2000; 57:838-845.-   Le Mellay V, et al., J Cell Biochem. 1999; 75:138-146.-   Leaney J L, Tinker A. Proc Natl Acad Sci U S A. 2000; 97:5651-5656.-   Li, P. A., et al., Neurosci. Lett. 177, 63-65 (1994).-   Lieberherr M, et al., J Cell Biochem. 1999; 74:50-60.-   Liss B, Roeper J. Mol Membr Biol. 2001; 18:117-127.-   Liu Y, et al., Circulation. 1998; 97:2463-2469.-   Mateo J, et al., Biochem J. 2003; 376:537-544.-   Mathews et al., J Neurosci Methods. 2000; 102:43-51.-   McNally J G, et al., Methods. 1999; 19:373-385.-   Meyer, M., et al., Br. J. Pharmacol. 128, 27-34 (1999).-   Moon R C, Constantinou AI. Breast Cancer Res Treat. 1997;    46:181-189.-   Moyer et al., J Neurosci Methods. 1998; 86:35-54.-   Munoz A, et al., Stroke. 2003; 34:164-170.-   Murayama T, et al., J Cell Physiol. 1996; 169:448-454.-   Murphy K, et al., Mol Pharmacol. 2003;, in press.-   Nakabayashi, K. et al. AJNR Am. J. Neuroradiol. 18, 485-491 (1997).-   Nichols C G, et al., Science. 1996; 272:1785-1787.-   Oehmichen M, et al., Exp Toxicol Pathol. 2000; 52:348-352.-   Oehmichen M, et al., Neurotoxicology. 2001; 22:99-107.-   Olive P L, et al., Br J Cancer. 2000; 83:1525-1531.-   Paczynski R P, et al., Stroke. 2000; 31:1702-1708.-   Paech K, et al., Science. 1997; 277:1508-1510.-   Panten U, et al., Biochem Pharmacol. 1989; 38:1217-1229.-   Papadopoulos M C, et al., Mt Sinai J Med. 2002; 69:242-248.-   Perillan P R, et al., J Biol Chem. 2002; 277:1974-1980.-   Perillan P R, et al., Glia. 1999; 27:213-225.-   Perillan P R, et al., Glia. 2000; 31:181-192.-   Phillips M I, Zhang Y C. Methods Enzymol. 2000; 313:46-56.-   Piiper A, et al., Am J Physiol. 1997; 272:G135-G140.-   Pogue B W, et al., Radiat Res. 2001; 155:15-25.-   Proks P, et al., J Physiol. 1999; 514 (Pt 1):19-25.-   Qiu J, et al., J Neurosci. 2003; 23:9529-9540.-   Rama Rao K V, et al., J Neurosci Res. 2003; 74:891-897.-   Rama Rao K V, et al., Neuroreport. 2003; 14:2379-2382.-   Ramirez V D, Zheng J. Front Neuroendocrinol. 1996; 17:402-439.-   Raucher D, et al., Cell. 2000; 100:221-228.-   Robinson A P, et al., Immunology. 1986; 57:239-247.-   Robinson S P, et al., Eur J Cancer Clin Oncol. 1988; 24:1817-1821.-   Rohacs T, et al., J Biol Chem. 1999; 274:36065-36072.-   Rossignol F, et al., Gene. 2002; 299:135-140.-   Ruknudin A, et al., J Biol Chem. 1998; 273:14165-14171.-   Ruscher K, et al., J Neurosci. 2002; 22:10291-10301.-   Russo J, et al., IARC Sci Publ. 1990; 47-78.-   Russo J, Russo I H. Lab Invest. 1987; 57:112-137.-   Saadoun S, et al., Br J Cancer. 2002; 87:621-623.-   Schubert P, et al., Ann N Y Acad Sci. 2000; 903:24-33.-   Seidel et al., Cell Tissue Res. 1991; 265:579-587.-   Seino, S. Annu. Rev. Physiol 61, 337-362 (1999).-   Semenza G L. Biochem Pharmacol. 2000; 59:47-53.-   Shaywitz B A, et al., Nature. 1995; 373:607-609.-   Shyng S, et al., J Gen Physiol. 1997; 110:643-654.-   Singer C A, et al., J Neurosci. 1999; 19:2455-2463.-   Singh M, et al., J Neurosci. 1999; 19:1179-1188.-   Smith S S, et al., Brain Res. 1987; 422:40-51.-   Smith S S, et al., Brain Res. 1988; 475:272-282.-   Sohrabji F, et al., Proc Natl Acad Sci U S A. 1995; 92:11110-11114.-   Stone D J, et al., J Neurosci. 1998; 18:3180-3185.-   Streit W J, et al., Frog Neurobiol. 1999; 57:563-581.-   Sun M C, et al., J Neurosurg. 2003; 98:565-569.-   Sylvia V L, et al, J Steroid Biochem Mol Biol. 2000; 73:211-224.-   Teixeira C, et al., Cancer Res. 1995; 55:3902-3907.-   Thrash-Bingham C A, et al., J Natl Cancer Inst. 1999; 91:143-151.-   Toker A. Curr Opin Cell Biol. 1998; 10:254-261.-   Toomey, J. R. et al. Stroke 33, 578-585 (2002).-   Toran-Allerand C D. J Steroid Biochem Mol Biol. 1996; 56:169-178.-   Torner L, et al., J Neurosci. 2001; 21:3207-3214.-   Treherne, J. M. & Ashford, M. L. Neuroscience 40, 523-531 (1991).-   Tucker S J, et al., EMBO J. 1998; 17:3290-3296.-   Tucker S J, et al., Nature. 1997; 387:179-183.-   U.S. Pat. No. 5,637,085-   U.S. Pat. No. 6,391,911-   Vogel et al., Stroke. 1999; 30:1134-1141.-   Wallace W, et al., Biotechniques. 2001; 31:1076-8, 1080, 1082.-   Wang J Y, et al., Glia. 2000; 32:155-164.-   Wang Y L. Methods Cell Biol. 1998; 56:305-315.-   Wass, C. T. & Lanier, W. L. Mayo Clin. Proc. 71, 801-812 (1996).-   Wiesener M S, et al., FASEB J. 2003; 17:271-273.-   WO 03/079987-   Woolley C S. Curr Opin Neurobiol. 1999; 9:349-354.-   Xie L H, et al., Proc Natl Acad Sci U S A. 1999; 96:15292-15297.-   Yajima Y, et al., Endocrinology. 1997; 138:1949-1958.-   Young, W. & Constantini, S. The Neurobiology of Central Nervous    System Trauma. Salzman, S.-   K. & Faden, A. I. (eds.), pp. 123-130 (Oxford University Press, New    York,1994).-   Zhang L, et al., Brain Res Mol Brain Res. 2002; 103:1-11.-   Zhang Y, et al., J Neurosci. 2001; 21:RC176.-   Zheng J, Ramirez V D. J Steroid Biochem Mol Biol. 1997; 62:327-336.-   Zunkler, B. J., et al., Biochem. Pharmacol. 67, 1437-1444 (2004).

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of reducing disruption of blood brain barrier in a subject in need thereof comprising administering glibenclamide or a pharmaceutically acceptable salt thereof and a thrombolytic agent to said subject.
 2. The method of claim 1, wherein the thrombolytic agent is a tissue plasminogen activator (tPA).
 3. The method of claim 1, wherein the thrombolytic agent is urokinase, prourokinase, streptokinase, anistreplase, reteplase, or tenecteplase.
 4. The method of claim 1, further comprising administering to the subject an anticoagulant or antiplatelet.
 5. The method of claim 4, wherein the anticoagulant or antiplatelet is aspirin, warfarin or coumadin.
 6. The method of claim 1, further comprising administering to the subject one or more statins, diuretics, or vasodilators.
 7. The method of claim 1, further comprising administering mannitol to the subject.
 8. The method of claim 1, wherein the glibenclamide or pharmaceutically acceptable salt thereof is administered alimentarily.
 9. The method of claim 8, wherein alimentarily comprises orally, buccally, rectally, or sublingually.
 10. The method of claim 1, wherein the glibenclamide or pharmaceutically acceptable salt thereof is administered parenterally.
 11. The method of claim 10, wherein parenterally comprises intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, intraperitoneally, or intraventricularly.
 12. The method of claim 10, wherein parenterally comprises injection into the brain parenchyma.
 13. The method of claim 1, wherein the glibenclamide or pharmaceutically acceptable salt thereof is administered at a dosage of 0.001 μg/kg/day to 100 μg/kg/day.
 14. The method of claim 1, wherein the glibenclamide or pharmaceutically acceptable salt thereof is administered at a loading dosage of 0.1 μg/kg to 100 μg/kg.
 15. The method of claim 1, wherein release of matrix metalloproteinase-2 and/or matrix metalloproteinase-9 from brain tissue of said subject is reduced. 